To read an article, click on its title and select the PDF file.
Use the box below to search publications.
2017 |
Strauch, Eva-Maria ; Bernard, Steffen M; La, David ; Bohn, Alan J; Lee, Peter S; Anderson, Caitlin E; Nieusma, Travis ; Holstein, Carly A; Garcia, Natalie K; Hooper, Kathryn A; Ravichandran, Rashmi ; Nelson, Jorgen W; Sheffler, William ; Bloom, Jesse D; Lee, Kelly K; Ward, Andrew B; Yager, Paul ; Fuller, Deborah H; Wilson, Ian A; Baker, David Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site Journal Article Nature Biotechnology, [Epub ahead of print] , 2017, ISSN: 1546-1696. @article{Strauch2017, title = {Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site}, author = {Strauch, Eva-Maria and Bernard, Steffen M and La, David and Bohn, Alan J and Lee, Peter S and Anderson, Caitlin E and Nieusma, Travis and Holstein, Carly A and Garcia, Natalie K and Hooper, Kathryn A and Ravichandran, Rashmi and Nelson, Jorgen W and Sheffler, William and Bloom, Jesse D and Lee, Kelly K and Ward, Andrew B and Yager, Paul and Fuller, Deborah H and Wilson, Ian A and Baker, David}, url = {https://www.bakerlab.org/wp-content/uploads/2017/06/Strauch_NatureBiotech_2017.pdf https://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3907.html}, doi = {10.1038/nbt.3907}, issn = {1546-1696}, year = {2017}, date = {2017-06-12}, journal = {Nature Biotechnology}, volume = {[Epub ahead of print]}, abstract = {Many viral surface glycoproteins and cell surface receptors are homo-oligomers, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza. }, keywords = {}, pubstate = {published}, tppubtype = {article} } Many viral surface glycoproteins and cell surface receptors are homo-oligomers, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza. |
CY, Janda; LT, Dang; C, You; J, Chang; de W, Lau; ZA, Zhong; KS, Yan; O, Marecic; D, Siepe; X, Li; JD, Moody; BO, Williams; H, Clevers; J, Piehler; D, Baker; CJ, Kuo; KC, Garcia Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling. Journal Article Nature, 545 (7653), pp. 234-237, 2017. @article{1001, title = {Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling.}, author = {Janda CY and Dang LT and You C and Chang J and de Lau W and Zhong ZA and Yan KS and Marecic O and Siepe D and Li X and Moody JD and Williams BO and Clevers H and Piehler J and Baker D and Kuo CJ and Garcia KC}, url = {https://www.bakerlab.org/wp-content/uploads/2018/06/nature22306.pdf http://www.nature.com/nature/journal/v545/n7653/abs/nature22306.html?foxtrotcallback=true}, doi = {10.1038/nature22306}, year = {2017}, date = {2017-05-11}, journal = {Nature}, volume = {545}, number = {7653}, pages = {234-237}, abstract = {Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues1. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors2, 3, 4. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD–LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues1. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors2, 3, 4. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD–LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine. |
Marcos, Enrique* ; Basanta, Benjamin* ; Chidyausiku, Tamuka M; Tang, Yuefeng ; Oberdorfer, Gustav ; Liu, Gaohua ; Swapna, G V T; Guan, Rongjin ; Silva, Daniel-Adriano ; Dou, Jiayi ; Pereira, Jose Henrique ; Xiao, Rong ; Sankaran, Banumathi ; Zwart, Peter H; Montelione, Gaetano T; Baker, David Principles for designing proteins with cavities formed by curved β sheets Journal Article Science, 355 (6321), pp. 201–206, 2017, ISSN: 0036-8075. @article{Marcos2017, title = {Principles for designing proteins with cavities formed by curved β sheets}, author = {Marcos, Enrique* and Basanta, Benjamin* and Chidyausiku, Tamuka M. and Tang, Yuefeng and Oberdorfer, Gustav and Liu, Gaohua and Swapna, G. V. T. and Guan, Rongjin and Silva, Daniel-Adriano and Dou, Jiayi and Pereira, Jose Henrique and Xiao, Rong and Sankaran, Banumathi and Zwart, Peter H. and Montelione, Gaetano T. and Baker, David}, url = {https://www.bakerlab.org/wp-content/uploads/2017/01/Marcos_Science_2017.pdf http://science.sciencemag.org/content/355/6321/201}, doi = {10.1126/science.aah7389}, issn = {0036-8075}, year = {2017}, date = {2017-01-01}, journal = {Science}, volume = {355}, number = {6321}, pages = {201--206}, publisher = {American Association for the Advancement of Science}, abstract = {In de novo protein design, creating custom-tailored binding sites is a particular challenge because these sites often involve nonideal backbone structures. For example, curved b sheets are a common ligand binding motif. Marcos et al. investigated the principles that drive β-sheet curvature by studying the geometry of β sheets in natural proteins and folding simulations. In a step toward custom design of enzyme catalysts, they used these principles to control β-sheet geometry and design proteins with differently shaped cavities.Science, this issue p. 201Active sites and ligand-binding cavities in native proteins are often formed by curved β sheets, and the ability to control β-sheet curvature would allow design of binding proteins with cavities customized to specific ligands. Toward this end, we investigated the mechanisms controlling β-sheet curvature by studying the geometry of β sheets in naturally occurring protein structures and folding simulations. The principles emerging from this analysis were used to design, de novo, a series of proteins with curved β sheets topped with α helices. Nuclear magnetic resonance and crystal structures of the designs closely match the computational models, showing that β-sheet curvature can be controlled with atomic-level accuracy. Our approach enables the design of proteins with cavities and provides a route to custom design ligand-binding and catalytic sites.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In de novo protein design, creating custom-tailored binding sites is a particular challenge because these sites often involve nonideal backbone structures. For example, curved b sheets are a common ligand binding motif. Marcos et al. investigated the principles that drive β-sheet curvature by studying the geometry of β sheets in natural proteins and folding simulations. In a step toward custom design of enzyme catalysts, they used these principles to control β-sheet geometry and design proteins with differently shaped cavities.Science, this issue p. 201Active sites and ligand-binding cavities in native proteins are often formed by curved β sheets, and the ability to control β-sheet curvature would allow design of binding proteins with cavities customized to specific ligands. Toward this end, we investigated the mechanisms controlling β-sheet curvature by studying the geometry of β sheets in naturally occurring protein structures and folding simulations. The principles emerging from this analysis were used to design, de novo, a series of proteins with curved β sheets topped with α helices. Nuclear magnetic resonance and crystal structures of the designs closely match the computational models, showing that β-sheet curvature can be controlled with atomic-level accuracy. Our approach enables the design of proteins with cavities and provides a route to custom design ligand-binding and catalytic sites. |
Ovchinnikov, Sergey; Park, Hahnbeom; Varghese, Neha; Huang, Po-Ssu; Pavlopoulos, Georgios A; Kim, David E; Kamisetty, Hetunandan; Kyrpides, Nikos C; Baker, David Protein structure determination using metagenome sequence data Journal Article Science, 355 (6322), pp. 294–298, 2017, ISSN: 0036-8075. @article{Ovchinnikov294, title = {Protein structure determination using metagenome sequence data}, author = { Sergey Ovchinnikov and Hahnbeom Park and Neha Varghese and Po-Ssu Huang and Georgios A. Pavlopoulos and David E. Kim and Hetunandan Kamisetty and Nikos C. Kyrpides and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2017/01/ovchinnikov_science_2017.pdf http://science.sciencemag.org/content/355/6322/294}, doi = {10.1126/science.aah4043}, issn = {0036-8075}, year = {2017}, date = {2017-01-01}, journal = {Science}, volume = {355}, number = {6322}, pages = {294--298}, publisher = {American Association for the Advancement of Science}, abstract = {Fewer than a third of the 14,849 known protein families have at least one member with an experimentally determined structure. This leaves more than 5000 protein families with no structural information. Protein modeling using residue-residue contacts inferred from evolutionary data has been successful in modeling unknown structures, but it requires large numbers of aligned sequences. Ovchinnikov et al. augmented such sequence alignments with metagenome sequence data (see the Perspective by S"oding). They determined the number of sequences required to allow modeling, developed criteria for model quality, and, where possible, improved modeling by matching predicted contacts to known structures. Their method predicted quality structural models for 614 protein families, of which about 140 represent newly discovered protein folds.Science, this issue p. 294; see also p. 248Despite decades of work by structural biologists, there are still ~5200 protein families with unknown structure outside the range of comparative modeling. We show that Rosetta structure prediction guided by residue-residue contacts inferred from evolutionary information can accurately model proteins that belong to large families and that metagenome sequence data more than triple the number of protein families with sufficient sequences for accurate modeling. We then integrate metagenome data, contact-based structure matching, and Rosetta structure calculations to generate models for 614 protein families with currently unknown structures; 206 are membrane proteins and 137 have folds not represented in the Protein Data Bank. This approach provides the representative models for large protein families originally envisioned as the goal of the Protein Structure Initiative at a fraction of the cost.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Fewer than a third of the 14,849 known protein families have at least one member with an experimentally determined structure. This leaves more than 5000 protein families with no structural information. Protein modeling using residue-residue contacts inferred from evolutionary data has been successful in modeling unknown structures, but it requires large numbers of aligned sequences. Ovchinnikov et al. augmented such sequence alignments with metagenome sequence data (see the Perspective by S"oding). They determined the number of sequences required to allow modeling, developed criteria for model quality, and, where possible, improved modeling by matching predicted contacts to known structures. Their method predicted quality structural models for 614 protein families, of which about 140 represent newly discovered protein folds.Science, this issue p. 294; see also p. 248Despite decades of work by structural biologists, there are still ~5200 protein families with unknown structure outside the range of comparative modeling. We show that Rosetta structure prediction guided by residue-residue contacts inferred from evolutionary information can accurately model proteins that belong to large families and that metagenome sequence data more than triple the number of protein families with sufficient sequences for accurate modeling. We then integrate metagenome data, contact-based structure matching, and Rosetta structure calculations to generate models for 614 protein families with currently unknown structures; 206 are membrane proteins and 137 have folds not represented in the Protein Data Bank. This approach provides the representative models for large protein families originally envisioned as the goal of the Protein Structure Initiative at a fraction of the cost. |
2016 |
Mills, Jeremy H; Sheffler, William; Ener, Maraia E; Almhjell, Patrick J; Oberdorfer, Gustav; Pereira, José Henrique; Parmeggiani, Fabio; Sankaran, Banumathi; Zwart, Peter H; Baker, David Computational design of a homotrimeric metalloprotein with a trisbipyridyl core Journal Article PNAS, 113 (52), pp. 15012-15017, 2016. @article{1300, title = {Computational design of a homotrimeric metalloprotein with a trisbipyridyl core}, author = {Jeremy H. Mills and William Sheffler and Maraia E. Ener and Patrick J. Almhjell and Gustav Oberdorfer and José Henrique Pereira and Fabio Parmeggiani and Banumathi Sankaran and Peter H. Zwart and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2018/06/15012.full_.pdf http://www.pnas.org/content/113/52/15012.abstract }, doi = {10.1073/pnas.1600188113}, year = {2016}, date = {2016-12-08}, journal = {PNAS}, volume = {113}, number = {52}, pages = {15012-15017}, abstract = {Metal-chelating heteroaryl small molecules have found widespread use as building blocks for coordination-driven, self-assembling nanostructures. The metal-chelating noncanonical amino acid (2,2′-bipyridin-5yl)alanine (Bpy-ala) could, in principle, be used to nucleate specific metalloprotein assemblies if introduced into proteins such that one assembly had much lower free energy than all alternatives. Here we describe the use of the Rosetta computational methodology to design a self-assembling homotrimeric protein with [Fe(Bpy-ala)3]2+ complexes at the interface between monomers. X-ray crystallographic analysis of the homotrimer showed that the design process had near-atomic-level accuracy: The all-atom rmsd between the design model and crystal structure for the residues at the protein interface is ∼1.4 Å. These results demonstrate that computational protein design together with genetically encoded noncanonical amino acids can be used to drive formation of precisely specified metal-mediated protein assemblies that could find use in a wide range of photophysical applications.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Metal-chelating heteroaryl small molecules have found widespread use as building blocks for coordination-driven, self-assembling nanostructures. The metal-chelating noncanonical amino acid (2,2′-bipyridin-5yl)alanine (Bpy-ala) could, in principle, be used to nucleate specific metalloprotein assemblies if introduced into proteins such that one assembly had much lower free energy than all alternatives. Here we describe the use of the Rosetta computational methodology to design a self-assembling homotrimeric protein with [Fe(Bpy-ala)3]2+ complexes at the interface between monomers. X-ray crystallographic analysis of the homotrimer showed that the design process had near-atomic-level accuracy: The all-atom rmsd between the design model and crystal structure for the residues at the protein interface is ∼1.4 Å. These results demonstrate that computational protein design together with genetically encoded noncanonical amino acids can be used to drive formation of precisely specified metal-mediated protein assemblies that could find use in a wide range of photophysical applications. |
JA, Fallas; G, Ueda; W, Sheffler; V, Nguyen; DE, McNamara; B, Sankaran; JH, Pereira; F, Parmeggiani; TJ, Brunette; D, Cascio; TR, Yeates; P, Zwart; D, Baker Computational design of self-assembling cyclic protein homo-oligomers Journal Article Nature Chemistry, 9 , pp. 353–360, 2016. @article{Fallas2016, title = {Computational design of self-assembling cyclic protein homo-oligomers}, author = {Fallas JA and Ueda G and Sheffler W and Nguyen V and McNamara DE and Sankaran B and Pereira JH and Parmeggiani F and Brunette TJ and Cascio D and Yeates TR and Zwart P and Baker D}, url = {https://www.nature.com/articles/nchem.2673 https://www.bakerlab.org/wp-content/uploads/2020/10/Fassas-et-al-2016-Homooligomers.pdf}, doi = {10.1038/nchem.2673}, year = {2016}, date = {2016-12-05}, journal = {Nature Chemistry}, volume = {9}, pages = {353–360}, abstract = {Self-assembling cyclic protein homo-oligomers play important roles in biology, and the ability to generate custom homo-oligomeric structures could enable new approaches to probe biological function. Here we report a general approach to design cyclic homo-oligomers that employs a new residue-pair-transform method to assess the designability of a protein–protein interface. This method is sufficiently rapid to enable the systematic enumeration of cyclically docked arrangements of a monomer followed by sequence design of the newly formed interfaces. We use this method to design interfaces onto idealized repeat proteins that direct their assembly into complexes that possess cyclic symmetry. Of 96 designs that were characterized experimentally, 21 were found to form stable monodisperse homo-oligomers in solution, and 15 (four homodimers, six homotrimers, six homotetramers and one homopentamer) had solution small-angle X-ray scattering data consistent with the design models. X-ray crystal structures were obtained for five of the designs and each is very close to their corresponding computational model.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Self-assembling cyclic protein homo-oligomers play important roles in biology, and the ability to generate custom homo-oligomeric structures could enable new approaches to probe biological function. Here we report a general approach to design cyclic homo-oligomers that employs a new residue-pair-transform method to assess the designability of a protein–protein interface. This method is sufficiently rapid to enable the systematic enumeration of cyclically docked arrangements of a monomer followed by sequence design of the newly formed interfaces. We use this method to design interfaces onto idealized repeat proteins that direct their assembly into complexes that possess cyclic symmetry. Of 96 designs that were characterized experimentally, 21 were found to form stable monodisperse homo-oligomers in solution, and 15 (four homodimers, six homotrimers, six homotetramers and one homopentamer) had solution small-angle X-ray scattering data consistent with the design models. X-ray crystal structures were obtained for five of the designs and each is very close to their corresponding computational model. |
Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; and Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, Douglas W; Hockenbery, David M; Baker, David Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer Journal Article Elife, 2016. @article{S2016, title = {Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer}, author = {Stephanie Berger and Erik Procko and Daciana Margineantu and Erinna F Lee and Betty W Shen and Alex Zelter and Daniel-Adriano Silva and and Kusum Chawla and Marco J Herold and Jean-Marc Garnier and Richard Johnson and Michael J MacCoss and Guillaume Lessene and Trisha N Davis and Patrick S Stayton and Barry L Stoddard and W Douglas Fairlie and David M Hockenbery and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2017/01/Berger_elife_2016.pdf https://elifesciences.org/articles/20352}, doi = {10.7554/eLife.20352}, year = {2016}, date = {2016-11-02}, journal = {Elife}, abstract = {Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes. |
Huang, Po-Ssu; Boyken, Scott E; Baker, David The coming of age of de novo protein design Journal Article Nature, 537 , pp. 320-327, 2016. @article{Huang2016, title = {The coming of age of de novo protein design}, author = {Po-Ssu Huang and Scott E. Boyken and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/09/HuangBoyken_DeNovoDesign_Nature2016.pdf}, doi = {10.1038/nature19946}, year = {2016}, date = {2016-09-15}, journal = {Nature}, volume = {537}, pages = {320-327}, abstract = {There are 20200 possible amino-acid sequences for a 200-residue protein, of which the natural evolutionary process has sampled only an infinitesimal subset. De novo protein design explores the full sequence space, guided by the physical principles that underlie protein folding. Computational methodology has advanced to the point that a wide range of structures can be designed from scratch with atomic-level accuracy. Almost all protein engineering so far has involved the modification of naturally occurring proteins; it should now be possible to design new functional proteins from the ground up to tackle current challenges in biomedicine and nanotechnology.}, keywords = {}, pubstate = {published}, tppubtype = {article} } There are 20200 possible amino-acid sequences for a 200-residue protein, of which the natural evolutionary process has sampled only an infinitesimal subset. De novo protein design explores the full sequence space, guided by the physical principles that underlie protein folding. Computational methodology has advanced to the point that a wide range of structures can be designed from scratch with atomic-level accuracy. Almost all protein engineering so far has involved the modification of naturally occurring proteins; it should now be possible to design new functional proteins from the ground up to tackle current challenges in biomedicine and nanotechnology. |
Bhardwaj*, Gaurav; Mulligan*, Vikram Khipple; Bahl*, Christopher D; Gilmore, Jason M; Harvey, Peta J; Cheneval, Olivier; Buchko, Garry W; Pulavarti, Surya V S R K; Kaas, Quentin; Eletsky, Alexander; Huang, Po-Ssu; Johnsen, William A; Greisen, Per Jr; Rocklin, Gabriel J; Song, Yifan; Linsky, Thomas W; Watkins, Andrew; Rettie, Stephen A; Xianzhong Xu, Lauren Carter P; Bonneau, Richard; Olson, James M; Coutsias, Evangelos; Correnti, Colin E; Szyperski, Thomas; Craik, David J; Baker, David Accurate de novo design of hyperstable constrained peptides Journal Article Nature, 2016. @article{Bhardwaj2016, title = {Accurate de novo design of hyperstable constrained peptides}, author = { Gaurav Bhardwaj* and Vikram Khipple Mulligan* and Christopher D. Bahl* and Jason M. Gilmore and Peta J. Harvey and Olivier Cheneval and Garry W. Buchko and Surya V. S. R. K. Pulavarti and Quentin Kaas and Alexander Eletsky and Po-Ssu Huang and William A. Johnsen and Per Jr Greisen and Gabriel J. Rocklin and Yifan Song and Thomas W. Linsky and Andrew Watkins and Stephen A. Rettie and Xianzhong Xu, Lauren P. Carter and Richard Bonneau and James M. Olson and Evangelos Coutsias and Colin E. Correnti and Thomas Szyperski and David J. Craik and David Baker }, url = {https://www.bakerlab.org/wp-content/uploads/2016/09/Bhardwaj_Nature_2016.pdf}, doi = {10.1038/nature19791}, year = {2016}, date = {2016-09-14}, journal = {Nature}, abstract = {Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18–47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N–C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18–47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N–C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs. |
Bale, Jacob B; Gonen, Shane; Liu, Yuxi; Sheffler, William; Ellis, Daniel; Thomas, Chantz; Cascio, Duilio; Yeates, Todd O; Gonen, Tamir; King, Neil P; Baker, David Accurate design of megadalton-scale two-component icosahedral protein complexes Journal Article Science, 353 (6297), pp. 389-394, 2016. @article{Bale2016, title = {Accurate design of megadalton-scale two-component icosahedral protein complexes}, author = {Jacob B. Bale and Shane Gonen and Yuxi Liu and William Sheffler and Daniel Ellis and Chantz Thomas and Duilio Cascio and Todd O. Yeates and Tamir Gonen and Neil P. King and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/07/Bale_Science_2016.pdf}, doi = {10.1126/science.aaf8818}, year = {2016}, date = {2016-07-22}, journal = {Science}, volume = {353}, number = {6297}, pages = {389-394}, abstract = {Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines. |
Hsia*, Yang; Bale*, Jacob B; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K; Nattermann, ; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N; Gonen, Tamir; King, Neil P; Baker, David Design of a hyperstable 60-subunit protein icosahedron Journal Article Nature, 2016. @article{Hsia2016, title = {Design of a hyperstable 60-subunit protein icosahedron}, author = { Yang Hsia* and Jacob B. Bale* and Shane Gonen and Dan Shi and William Sheffler and Kimberly K. Fong and Nattermann and Chunfu Xu and Po-Ssu Huang and Rashmi Ravichandran and Sue Yi and Trisha N. Davis and Tamir Gonen and Neil P. King and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/Hsia_Nature_2016.pdf}, doi = {10.1038/nature18010}, year = {2016}, date = {2016-06-15}, journal = {Nature}, abstract = {The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/ exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/ exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology. |
Klein J. C., Lajoie Schwartz Strauch Nelson Baker & Shendure M J J J E -M J D J Multiplex pairwise assembly of array-derived DNA oligonucleotides Journal Article Nucleic Acids Research, 44 (5), pp. e43, 2016. @article{Klein2016, title = {Multiplex pairwise assembly of array-derived DNA oligonucleotides}, author = {Klein, J. C., Lajoie, M. J., Schwartz, J. J., Strauch, E.-M., Nelson, J., Baker, D., & Shendure, J}, url = {https://www.bakerlab.org/wp-content/uploads/2016/05/gkv1177.pdf}, doi = {10.1093/nar/gkv1177}, year = {2016}, date = {2016-03-18}, journal = {Nucleic Acids Research}, volume = {44}, number = {5}, pages = {e43}, abstract = {While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192–252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131–250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput ‘Dial-Out PCR’ protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.}, keywords = {}, pubstate = {published}, tppubtype = {article} } While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192–252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131–250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput ‘Dial-Out PCR’ protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens. |
Taylor ND Garruss AS, Moretti Chan Arbing MA Cascio Rogers JK Isaacs FJ Kosuri Baker Fields Church GM Raman R S D S D S S Engineering an allosteric transcription factor to respond to new ligands Journal Article Nature Methods, 13 (2), pp. 177-83, 2016. @article{ND2016, title = {Engineering an allosteric transcription factor to respond to new ligands}, author = {Taylor ND, Garruss AS, Moretti R, Chan S, Arbing MA, Cascio D, Rogers JK, Isaacs FJ, Kosuri S, Baker D, Fields S, Church GM, Raman S}, url = {https://www.bakerlab.org/wp-content/uploads/2016/05/nmeth.36961.pdf}, doi = {10.1038/nmeth.3696}, year = {2016}, date = {2016-02-01}, journal = {Nature Methods}, volume = {13}, number = {2}, pages = {177-83}, abstract = {Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits}, keywords = {}, pubstate = {published}, tppubtype = {article} } Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits |
Boyken, Scott E; Chen, Zibo; Groves, Benjamin; Langan, Robert A; Oberdorfer, Gustav; Ford, Alex; Gilmore, Jason M; Xu, Chunfu; DiMaio, Frank; Pereira, Jose Henrique; Sankaran, Banumathi; Seelig, Georg; Zwart, Peter H; Baker, David De novo design of protein homo-oligomers with modular hydrogen-bond network–mediated specificity Journal Article Science, 352 (6286), pp. 680–687, 2016, ISSN: 0036-8075. @article{Boyken680, title = {De novo design of protein homo-oligomers with modular hydrogen-bond network–mediated specificity}, author = { Scott E. Boyken and Zibo Chen and Benjamin Groves and Robert A. Langan and Gustav Oberdorfer and Alex Ford and Jason M. Gilmore and Chunfu Xu and Frank DiMaio and Jose Henrique Pereira and Banumathi Sankaran and Georg Seelig and Peter H. Zwart and David Baker}, url = {http://science.sciencemag.org/content/352/6286/680 https://www.bakerlab.org/wp-content/uploads/2016/05/680.full_.pdf}, doi = {10.1126/science.aad8865}, issn = {0036-8075}, year = {2016}, date = {2016-01-01}, journal = {Science}, volume = {352}, number = {6286}, pages = {680--687}, publisher = {American Association for the Advancement of Science}, abstract = {General design principles for protein interaction specificity are challenging to extract. DNA nanotechnology, on the other hand, has harnessed the limited set of hydrogen-bonding interactions from Watson-Crick base-pairing to design and build a wide range of shapes. Protein-based materials have the potential for even greater geometric and chemical diversity, including additional functionality. Boyken et al. designed a class of protein oligomers that have interaction specificity determined by modular arrays of extensive hydrogen bond networks (see the Perspective by Netzer and Fleishman). They use the approach, which could one day become programmable, to build novel topologies with two concentric rings of helices.Science, this issue p. 680; see also p. 657In nature, structural specificity in DNA and proteins is encoded differently: In DNA, specificity arises from modular hydrogen bonds in the core of the double helix, whereas in proteins, specificity arises largely from buried hydrophobic packing complemented by irregular peripheral polar interactions. Here, we describe a general approach for designing a wide range of protein homo-oligomers with specificity determined by modular arrays of central hydrogen-bond networks. We use the approach to design dimers, trimers, and tetramers consisting of two concentric rings of helices, including previously not seen triangular, square, and supercoiled topologies. X-ray crystallography confirms that the structures overall, and the hydrogen-bond networks in particular, are nearly identical to the design models, and the networks confer interaction specificity in vivo. The ability to design extensive hydrogen-bond networks with atomic accuracy enables the programming of protein interaction specificity for a broad range of synthetic biology applications; more generally, our results demonstrate that, even with the tremendous diversity observed in nature, there are fundamentally new modes of interaction to be discovered in proteins.}, keywords = {}, pubstate = {published}, tppubtype = {article} } General design principles for protein interaction specificity are challenging to extract. DNA nanotechnology, on the other hand, has harnessed the limited set of hydrogen-bonding interactions from Watson-Crick base-pairing to design and build a wide range of shapes. Protein-based materials have the potential for even greater geometric and chemical diversity, including additional functionality. Boyken et al. designed a class of protein oligomers that have interaction specificity determined by modular arrays of extensive hydrogen bond networks (see the Perspective by Netzer and Fleishman). They use the approach, which could one day become programmable, to build novel topologies with two concentric rings of helices.Science, this issue p. 680; see also p. 657In nature, structural specificity in DNA and proteins is encoded differently: In DNA, specificity arises from modular hydrogen bonds in the core of the double helix, whereas in proteins, specificity arises largely from buried hydrophobic packing complemented by irregular peripheral polar interactions. Here, we describe a general approach for designing a wide range of protein homo-oligomers with specificity determined by modular arrays of central hydrogen-bond networks. We use the approach to design dimers, trimers, and tetramers consisting of two concentric rings of helices, including previously not seen triangular, square, and supercoiled topologies. X-ray crystallography confirms that the structures overall, and the hydrogen-bond networks in particular, are nearly identical to the design models, and the networks confer interaction specificity in vivo. The ability to design extensive hydrogen-bond networks with atomic accuracy enables the programming of protein interaction specificity for a broad range of synthetic biology applications; more generally, our results demonstrate that, even with the tremendous diversity observed in nature, there are fundamentally new modes of interaction to be discovered in proteins. |
Ovchinnikov, Sergey ; Park, Hahnbeom ; Kim, David E; Liu, Yuan ; Wang, Ray Yu-Ruei ; Baker, David Structure prediction using sparse simulated NOE restraints with Rosetta in CASP11 Journal Article Proteins: Structure, Function, and Bioinformatics, pp. n/a–n/a, 2016, ISSN: 1097-0134. @article{PROT:PROT25006, title = {Structure prediction using sparse simulated NOE restraints with Rosetta in CASP11}, author = {Ovchinnikov, Sergey and Park, Hahnbeom and Kim, David E. and Liu, Yuan and Wang, Ray Yu-Ruei and Baker, David}, url = {http://dx.doi.org/10.1002/prot.25006 https://www.bakerlab.org/wp-content/uploads/2016/05/Ovchinnikov_et_al-2016-Proteins__Structure_Function_and_Bioinformatics.pdf}, doi = {10.1002/prot.25006}, issn = {1097-0134}, year = {2016}, date = {2016-01-01}, journal = {Proteins: Structure, Function, and Bioinformatics}, pages = {n/a--n/a}, abstract = {In CASP11 we generated protein structure models using simulated ambiguous and unambiguous nuclear Overhauser effect (NOE) restraints with a two stage protocol. Low resolution models were generated guided by the unambiguous restraints using continuous chain folding for alpha and alpha-beta proteins, and iterative annealing for all beta proteins to take advantage of the strand pairing information implicit in the restraints. The Rosetta fragment/model hybridization protocol was then used to recombine and regularize these models, and refine them in the Rosetta full atom energy function guided by both the unambiguous and the ambiguous restraints. Fifteen out of 19 targets were modeled with GDT-TS quality scores greater than 60 for Model 1, significantly improving upon the non-assisted predictions. Our results suggest that atomic level accuracy is achievable using sparse NOE data when there is at least one correctly assigned NOE for every residue. Proteins 2016. © 2016 Wiley Periodicals, Inc.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In CASP11 we generated protein structure models using simulated ambiguous and unambiguous nuclear Overhauser effect (NOE) restraints with a two stage protocol. Low resolution models were generated guided by the unambiguous restraints using continuous chain folding for alpha and alpha-beta proteins, and iterative annealing for all beta proteins to take advantage of the strand pairing information implicit in the restraints. The Rosetta fragment/model hybridization protocol was then used to recombine and regularize these models, and refine them in the Rosetta full atom energy function guided by both the unambiguous and the ambiguous restraints. Fifteen out of 19 targets were modeled with GDT-TS quality scores greater than 60 for Model 1, significantly improving upon the non-assisted predictions. Our results suggest that atomic level accuracy is achievable using sparse NOE data when there is at least one correctly assigned NOE for every residue. Proteins 2016. © 2016 Wiley Periodicals, Inc. |
Basanta, Benjamin; Chan, Kui K; Barth, Patrick; King, Tiffany; Sosnick, Tobin R; Hinshaw, James R; Liu, Gaohua; Everett, John K; Xiao, Rong; Montelione, Gaetano T; Baker, David Introduction of a polar core into the de novo designed protein Top7 Journal Article Protein Science, pp. n/a–n/a, 2016, ISSN: 1469-896X. @article{PRO:PRO2899, title = {Introduction of a polar core into the de novo designed protein Top7}, author = { Benjamin Basanta and Kui K. Chan and Patrick Barth and Tiffany King and Tobin R. Sosnick and James R. Hinshaw and Gaohua Liu and John K. Everett and Rong Xiao and Gaetano T. Montelione and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/05/Basanta_et_al-2016-Protein_Science.pdf http://dx.doi.org/10.1002/pro.2899}, doi = {10.1002/pro.2899}, issn = {1469-896X}, year = {2016}, date = {2016-01-01}, journal = {Protein Science}, pages = {n/a--n/a}, abstract = {Design of polar interactions is a current challenge for protein design. The de novo designed protein Top7, like almost all designed proteins, has an entirely nonpolar core. Here we describe the replacing of a sizable fraction (5 residues) of this core with a designed polar hydrogen bond network. The polar core design is expressed at high levels in E. coli, has a folding free energy of 10 kcal/mol, and retains the multiphasic folding kinetics of the original Top7. The NMR structure of the design shows that conformations of three of the five residues, and the designed hydrogen bonds between them, are very close to those in the design model. The remaining two residues, which are more solvent exposed, sample a wide range of conformations in the NMR ensemble. These results show that hydrogen bond networks can be designed in protein cores, but also highlight challenges that need to be overcome when there is competition with solvent.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Design of polar interactions is a current challenge for protein design. The de novo designed protein Top7, like almost all designed proteins, has an entirely nonpolar core. Here we describe the replacing of a sizable fraction (5 residues) of this core with a designed polar hydrogen bond network. The polar core design is expressed at high levels in E. coli, has a folding free energy of 10 kcal/mol, and retains the multiphasic folding kinetics of the original Top7. The NMR structure of the design shows that conformations of three of the five residues, and the designed hydrogen bonds between them, are very close to those in the design model. The remaining two residues, which are more solvent exposed, sample a wide range of conformations in the NMR ensemble. These results show that hydrogen bond networks can be designed in protein cores, but also highlight challenges that need to be overcome when there is competition with solvent. |
Treants, Merika; Jorgen, Nelson; Aaron, Chevalier; Michael, Koday; Hannah, Kalinoski; Lance, Stewart; Lauren, Carter; Travis, Nieusma; S., Lee Peter; B., Ward Andrew; A., Wilson Ian; Ashley, Dagley; F., Smee Donald; David, Baker; Koday, Fuller Deborah Heydenburg A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response Journal Article PLoS Pathog, 12 (2), pp. 1-23, 2016. @article{10.1371/journal.ppat.1005409, title = {A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response}, author = { Merika Treants AND Nelson Jorgen AND Chevalier Aaron AND Koday Michael AND Kalinoski Hannah AND Stewart Lance AND Carter Lauren AND Nieusma Travis AND Lee Peter S. AND Ward Andrew B. AND Wilson Ian A. AND Dagley Ashley AND Smee Donald F. AND Baker David AND Fuller Deborah Heydenburg Koday}, url = {http://dx.doi.org/10.1371%2Fjournal.ppat.1005409 https://www.bakerlab.org/wp-content/uploads/2016/05/journal.ppat_.1005409.pdf}, doi = {10.1371/journal.ppat.1005409}, year = {2016}, date = {2016-01-01}, journal = {PLoS Pathog}, volume = {12}, number = {2}, pages = {1-23}, publisher = {Public Library of Science}, abstract = { Influenza is a major public health threat, and pandemics, such as the 2009 H1N1 outbreak, are inevitable. Due to low efficacy of seasonal flu vaccines and the increase in drug-resistant strains of influenza viruses, there is a crucial need to develop new antivirals to protect from seasonal and pandemic influenza. Recently, several broadly neutralizing antibodies have been characterized that bind to a highly conserved site on the viral hemagglutinin (HA) stem region. These antibodies are protective against a wide range of diverse influenza viruses, but their efficacy depends on a host immune effector response through the antibody Fc region (ADCC). Here we show that a small engineered protein computationally designed to bind to the same region of the HA stem as broadly neutralizing antibodies mediated protection against diverse strains of influenza in mice by a distinct mechanism that is independent of a host immune response. Protection was superior to that afforded by oseltamivir, a lead marketed antiviral. Furthermore, combination therapy with low doses of the engineered protein and oseltamivir resulted in enhanced and synergistic protection from lethal challenge. Thus, through computational protein engineering, we have designed a new antiviral with strong biopotency keywords = {}, pubstate = {published}, tppubtype = {article} } <title>Author Summary</title> <p>Influenza is a major public health threat, and pandemics, such as the 2009 H1N1 outbreak, are inevitable. Due to low efficacy of seasonal flu vaccines and the increase in drug-resistant strains of influenza viruses, there is a crucial need to develop new antivirals to protect from seasonal and pandemic influenza. Recently, several broadly neutralizing antibodies have been characterized that bind to a highly conserved site on the viral hemagglutinin (HA) stem region. These antibodies are protective against a wide range of diverse influenza viruses, but their efficacy depends on a host immune effector response through the antibody Fc region (ADCC). Here we show that a small engineered protein computationally designed to bind to the same region of the HA stem as broadly neutralizing antibodies mediated protection against diverse strains of influenza in mice by a distinct mechanism that is independent of a host immune response. Protection was superior to that afforded by oseltamivir, a lead marketed antiviral. Furthermore, combination therapy with low doses of the engineered protein and oseltamivir resulted in enhanced and synergistic protection from lethal challenge. Thus, through computational protein engineering, we have designed a new antiviral with strong biopotency <italic>in vivo</italic> that targets a neutralizing epitope on the hemagglutinin of influenza virus and inhibits its fusion activity. These results have significant implications for the use of computational modeling to design new antivirals against influenza and other viral diseases.</p> |
Garcia, Kristen E; Babanova, Sofia; Scheffler, William; Hans, Mansij; Baker, David; Atanassov, Plamen; Banta, Scott Designed protein aggregates entrapping carbon nanotubes for bioelectrochemical oxygen reduction Journal Article Biotechnology and Bioengineering, pp. n/a–n/a, 2016, ISSN: 1097-0290. @article{BIT:BIT25996, title = {Designed protein aggregates entrapping carbon nanotubes for bioelectrochemical oxygen reduction}, author = { Kristen E Garcia and Sofia Babanova and William Scheffler and Mansij Hans and David Baker and Plamen Atanassov and Scott Banta}, url = {http://dx.doi.org/10.1002/bit.25996 https://www.bakerlab.org/wp-content/uploads/2016/05/Garcia_et_al-2016-Biotechnology_and_Bioengineering.pdf}, doi = {10.1002/bit.25996}, issn = {1097-0290}, year = {2016}, date = {2016-01-01}, journal = {Biotechnology and Bioengineering}, pages = {n/a--n/a}, abstract = {The engineering of robust protein/nanomaterial interfaces is critical in the development of bioelectrocatalytic systems. We have used computational protein design to identify two amino acid mutations in the small laccase protein (SLAC) from Streptomyces coelicolor to introduce new inter-protein disulfide bonds. The new dimeric interface introduced by these disulfide bonds in combination with the natural trimeric structure drive the self-assembly of SLAC into functional aggregates. The mutations had a minimal effect on kinetic parameters, and the enzymatic assemblies exhibited an increased resistance to irreversible thermal denaturation. The SLAC assemblies were combined with single-walled carbon nanotubes (SWNTs), and explored for use in oxygen reduction electrodes. The incorporation of SWNTs into the SLAC aggregates enabled operation an elevated temperature and reduced the reaction overpotential. A current density of 1.1 mA/cm2 at 0 V vs. Ag/AgCl was achieved in an air-breathing cathode system. This article is protected by copyright. All rights reserved}, keywords = {}, pubstate = {published}, tppubtype = {article} } The engineering of robust protein/nanomaterial interfaces is critical in the development of bioelectrocatalytic systems. We have used computational protein design to identify two amino acid mutations in the small laccase protein (SLAC) from Streptomyces coelicolor to introduce new inter-protein disulfide bonds. The new dimeric interface introduced by these disulfide bonds in combination with the natural trimeric structure drive the self-assembly of SLAC into functional aggregates. The mutations had a minimal effect on kinetic parameters, and the enzymatic assemblies exhibited an increased resistance to irreversible thermal denaturation. The SLAC assemblies were combined with single-walled carbon nanotubes (SWNTs), and explored for use in oxygen reduction electrodes. The incorporation of SWNTs into the SLAC aggregates enabled operation an elevated temperature and reduced the reaction overpotential. A current density of 1.1 mA/cm2 at 0 V vs. Ag/AgCl was achieved in an air-breathing cathode system. This article is protected by copyright. All rights reserved |
2015 |
Feng, J; Jester, BW; Tinberg, CE; Mandell, DJ; Antunes, MS; Chari, R; Morey, KJ; Rios, X; Medford, JI; Church, GM; Fields, S; Baker, D A general strategy to construct small molecule biosensors in eukaryotes Journal Article Elife, 2015. @article{J2015, title = {A general strategy to construct small molecule biosensors in eukaryotes}, author = {J Feng and BW Jester and CE Tinberg and DJ Mandell and MS Antunes and R Chari and KJ Morey and X Rios and JI Medford and GM Church and S Fields and D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/04/elife-10606-v3-download.pdf}, doi = {10.7554/eLife.10606}, year = {2015}, date = {2015-12-29}, journal = {Elife}, abstract = {Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes. |
Doyle, L; Hallinan, J; Bolduc, J; Parmeggiani, F; Baker, D; Stoddard, BL; Bradley, P Rational design of α-helical tandem repeat proteins with closed architectures Journal Article Nature, 528(7583) , pp. 585-8, 2015. @article{L2015, title = {Rational design of α-helical tandem repeat proteins with closed architectures}, author = {L Doyle and J Hallinan and J Bolduc and F Parmeggiani and D Baker and BL Stoddard and P Bradley}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Doyle_Nature_2015.pdf}, doi = {10.1038/nature16191}, year = {2015}, date = {2015-12-24}, journal = {Nature}, volume = {528(7583)}, pages = {585-8}, abstract = {Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks. The overall architecture of tandem repeat protein structures--which is dictated by the internal geometry and local packing of the repeat building blocks--is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed α-solenoid repeat structures (α-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed α-solenoid repeats with a left-handed helical architecture that--to our knowledge--is not yet present in the protein structure database.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks. The overall architecture of tandem repeat protein structures--which is dictated by the internal geometry and local packing of the repeat building blocks--is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed α-solenoid repeat structures (α-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed α-solenoid repeats with a left-handed helical architecture that--to our knowledge--is not yet present in the protein structure database. |
Brunette, TJ; Parmeggiani, F; Huang, PS; Bhabha, G; Ekiert, DC; Tsutakawa, SE; Hura, GL; Tainer, JA; Baker, D Exploring the repeat protein universe through computational protein design Journal Article Nature, 528(7583) , pp. 580-4, 2015. @article{TJ2015, title = {Exploring the repeat protein universe through computational protein design}, author = {TJ Brunette and F Parmeggiani and PS Huang and G Bhabha and DC Ekiert and SE Tsutakawa and GL Hura and JA Tainer and D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Brunette_Nature_2015.pdf}, doi = {10.1038/nature16162}, year = {2015}, date = {2015-12-24}, journal = {Nature}, volume = {528(7583)}, pages = {580-4}, abstract = {A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering. }, keywords = {}, pubstate = {published}, tppubtype = {article} } A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering. |
Taylor, ND; Garruss, AS; Moretti, R; Chan, S; Arbing, MA; Cascio, D; Rogers, JK; Isaacs, FJ; Kosuri, S; Baker, D; Fields, S; Church, GM; Raman, S Engineering an allosteric transcription factor to respond to new ligands Journal Article Nature Methods, 2015. @article{ND2015, title = {Engineering an allosteric transcription factor to respond to new ligands}, author = {ND Taylor and AS Garruss and R Moretti and S Chan and MA Arbing and D Cascio and JK Rogers and FJ Isaacs and S Kosuri and D Baker and S Fields and GM Church and S Raman}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Taylor_NatMeth_2015.pdf}, doi = {10.1038/nmeth.3696}, year = {2015}, date = {2015-12-21}, journal = {Nature Methods}, abstract = {Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits. |
Ovchinnikov, S; Kim, DE; Wang, RY; Liu, Y; DiMaio, F; Baker, D Improved de novo structure prediction in CASP11 by incorporating Co-evolution information into rosetta Journal Article Proteins, 2015. @article{S2015, title = {Improved de novo structure prediction in CASP11 by incorporating Co-evolution information into rosetta}, author = {S Ovchinnikov and DE Kim and RY Wang and Y Liu and F DiMaio and D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Ovchinnikov_Proteins_2015.pdf}, doi = {10.1002/prot.24974}, year = {2015}, date = {2015-12-17}, journal = {Proteins}, abstract = {We describe CASP11 de novo blind structure predictions made using the Rosetta structure prediction methodology with both automatic and human assisted protocols. Model accuracy was generally improved using co-evolution derived residue-residue contact information as restraints during Rosetta conformational sampling and refinement, particularly when the number of sequences in the family was more than three times the length of the protein. The highlight was the human assisted prediction of T0806, a large and topologically complex target with no homologs of known structure, which had unprecedented accuracy - <3.0 Å root-mean-square deviation (RMSD) from the crystal structure over 223 residues. For this target, we increased the amount of conformational sampling over our fully automated method by employing an iterative hybridization protocol. Our results clearly demonstrate, in a blind prediction scenario, that co-evolution derived contacts can considerably increase the accuracy of template-free structure modeling. This article is protected by copyright. All rights reserved.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe CASP11 de novo blind structure predictions made using the Rosetta structure prediction methodology with both automatic and human assisted protocols. Model accuracy was generally improved using co-evolution derived residue-residue contact information as restraints during Rosetta conformational sampling and refinement, particularly when the number of sequences in the family was more than three times the length of the protein. The highlight was the human assisted prediction of T0806, a large and topologically complex target with no homologs of known structure, which had unprecedented accuracy - <3.0 Å root-mean-square deviation (RMSD) from the crystal structure over 223 residues. For this target, we increased the amount of conformational sampling over our fully automated method by employing an iterative hybridization protocol. Our results clearly demonstrate, in a blind prediction scenario, that co-evolution derived contacts can considerably increase the accuracy of template-free structure modeling. This article is protected by copyright. All rights reserved. |
King, IC; Gleixner, J; Doyle, L; Kuzin, A; Hunt, JF; Xiao, R; Montelione, GT; Stoddard, BL; DiMaio, F; Baker, D Precise assembly of complex beta sheet topologies from de novo designed building blocks Journal Article Elife, 2015. @article{IC2015, title = {Precise assembly of complex beta sheet topologies from de novo designed building blocks}, author = {IC King and J Gleixner and L Doyle and A Kuzin and JF Hunt and R Xiao and GT Montelione and BL Stoddard and F DiMaio and D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/King_elife_2015.pdf}, doi = {10.7554/eLife.11012}, year = {2015}, date = {2015-12-09}, journal = {Elife}, abstract = {Design of complex alpha-beta protein topologies poses a challenge because of the large number of alternative packing arrangements. A similar challenge presumably limited the emergence of large and complex protein topologies in evolution. Here we demonstrate that protein topologies with six and seven-stranded beta sheets can be designed by insertion of one de novo designed beta sheet containing protein into another such that the two beta sheets are merged to form a single extended sheet, followed by amino acid sequence optimization at the newly formed strand-strand, strand-helix, and helix-helix interfaces. Crystal structures of two such designs closely match the computational design models. Searches for similar structures in the SCOP protein domain database yield only weak matches with different beta sheet connectivities. A similar beta sheet fusion mechanism may have contributed to the emergence of complex beta sheets during natural protein evolution.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Design of complex alpha-beta protein topologies poses a challenge because of the large number of alternative packing arrangements. A similar challenge presumably limited the emergence of large and complex protein topologies in evolution. Here we demonstrate that protein topologies with six and seven-stranded beta sheets can be designed by insertion of one de novo designed beta sheet containing protein into another such that the two beta sheets are merged to form a single extended sheet, followed by amino acid sequence optimization at the newly formed strand-strand, strand-helix, and helix-helix interfaces. Crystal structures of two such designs closely match the computational design models. Searches for similar structures in the SCOP protein domain database yield only weak matches with different beta sheet connectivities. A similar beta sheet fusion mechanism may have contributed to the emergence of complex beta sheets during natural protein evolution. |
Goldsmith, M; Eckstein, S; Ashani, Y; Greisen, Jr P; Leader, H; Sussman, JL; Aggarwal, N; Ovchinnikov, S; Tawfik, DS; Baker, D; Thiermann, H; Worek, F Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro Journal Article Archives of Toxicology, 2015. @article{M2015, title = {Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro}, author = {M Goldsmith and S Eckstein and Y Ashani and P Jr Greisen and H Leader and JL Sussman and N Aggarwal and S Ovchinnikov and DS Tawfik and D Baker and H Thiermann and F Worek}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Goldsmith_ArchToxicol_2015.pdf}, doi = {10.1007/s00204-015-1626-2}, year = {2015}, date = {2015-11-26}, journal = {Archives of Toxicology}, abstract = {The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships. |
Huang, PS; Feldmeier, K; Parmeggiani, F; Velasco, DA Fernandez; Höcker, B; Baker, D De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy Journal Article Nature Chemical Biology, 12(1) , pp. 29-34, 2015. @article{PS2015, title = {De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy}, author = {PS Huang and K Feldmeier and F Parmeggiani and DA Fernandez Velasco and B Höcker and D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Huang_NatChemBio_2015.pdf}, doi = {10.1038/nchembio.1966}, year = {2015}, date = {2015-11-23}, journal = {Nature Chemical Biology}, volume = {12(1)}, pages = {29-34}, abstract = {Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of four-fold symmetrical (β/α)8 barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of side chain-backbone hydrogen bonds for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical to that of the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has sampled only a subset of the sequence space available to the TIM-barrel fold. The ability to design TIM barrels de novo opens new possibilities for custom-made enzymes. }, keywords = {}, pubstate = {published}, tppubtype = {article} } Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of four-fold symmetrical (β/α)8 barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of side chain-backbone hydrogen bonds for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical to that of the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has sampled only a subset of the sequence space available to the TIM-barrel fold. The ability to design TIM barrels de novo opens new possibilities for custom-made enzymes. |
Lin YR Koga N, Tatsumi-Koga Liu Clouser AF Montelione GT Baker R G D Control over overall shape and size in de novo designed proteins Journal Article Proc Natl Acad Sci U S A., pp. E5478-85, 2015. @article{YR2015, title = {Control over overall shape and size in de novo designed proteins}, author = {Lin YR, Koga N, Tatsumi-Koga R, Liu G, Clouser AF, Montelione GT, Baker D}, url = {https://www.bakerlab.org/wp-content/uploads/2016/04/PNAS-2015-Lin-E5478-85.pdf}, doi = {10.1073/pnas.1509508112}, year = {2015}, date = {2015-10-06}, journal = {Proc Natl Acad Sci U S A.}, pages = {E5478-85}, abstract = {We recently described general principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions. The principles relate secondary structure patterns to tertiary packing motifs and enable design of different protein topologies. To achieve fine control over protein shape and size within a particular topology, we have extended the design rules by systematically analyzing the codependencies between the lengths and packing geometry of successive secondary structure elements and the backbone torsion angles of the loop linking them. We demonstrate the control afforded by the resulting extended rule set by designing a series of proteins with the same fold but considerable variation in secondary structure length, loop geometry, β-strand registry, and overall shape. Solution NMR structures of four designed proteins for two different folds show that protein shape and size can be precisely controlled within a given protein fold. These extended design principles provide the foundation for custom design of protein structures performing desired functions. }, keywords = {}, pubstate = {published}, tppubtype = {article} } We recently described general principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions. The principles relate secondary structure patterns to tertiary packing motifs and enable design of different protein topologies. To achieve fine control over protein shape and size within a particular topology, we have extended the design rules by systematically analyzing the codependencies between the lengths and packing geometry of successive secondary structure elements and the backbone torsion angles of the loop linking them. We demonstrate the control afforded by the resulting extended rule set by designing a series of proteins with the same fold but considerable variation in secondary structure length, loop geometry, β-strand registry, and overall shape. Solution NMR structures of four designed proteins for two different folds show that protein shape and size can be precisely controlled within a given protein fold. These extended design principles provide the foundation for custom design of protein structures performing desired functions. |
Holstein, Carly A; Chevalier, Aaron; Bennett, Steven; Anderson, Caitlin E; Keniston, Karen; Olsen, Cathryn; Li, Bing; Bales, Brian; Moore, David R; Fu, Elain; Baker, David; Yager, Paul Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers. Journal Article Analytical and bioanalytical chemistry, 2015, ISSN: 1618-2650. @article{626, title = {Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.}, author = { Carly A Holstein and Aaron Chevalier and Steven Bennett and Caitlin E Anderson and Karen Keniston and Cathryn Olsen and Bing Li and Brian Bales and David R Moore and Elain Fu and David Baker and Paul Yager}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Holstien_Anal_Bioanal_Chem_2015.pdf}, doi = {10.1007/s00216-015-9052-0}, issn = {1618-2650}, year = {2015}, date = {2015-10-01}, journal = {Analytical and bioanalytical chemistry}, abstract = {To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.}, keywords = {}, pubstate = {published}, tppubtype = {article} } To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics. |
S Ovchinnikov L Kinch, Park Liao Pei DE Kim Kamisetty NV Grishin Baker H Y J H D Large-scale determination of previously unsolved protein structures using evolutionary information Journal Article eLife, 2015. @article{S2015b, title = {Large-scale determination of previously unsolved protein structures using evolutionary information}, author = {S Ovchinnikov, L Kinch, H Park, Y Liao, J Pei, DE Kim, H Kamisetty, NV Grishin, D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/01/Ovchinnikov_eLife_2015.pdf}, doi = {10.7554/eLife.09248}, year = {2015}, date = {2015-09-03}, journal = {eLife}, abstract = {The prediction of the structures of proteins without detectable sequence similarity to any protein of known structure remains an outstanding scientific challenge. Here we report significant progress in this area. We first describe de novo blind structure predictions of unprecendented accuracy we made for two proteins in large families in the recent CASP11 blind test of protein structure prediction methods by incorporating residue-residue co-evolution information in the Rosetta structure prediction program. We then describe the use of this method to generate structure models for 58 of the 121 large protein families in prokaryotes for which three-dimensional structures are not available. These models, which are posted online for public access, provide structural information for the over 400,000 proteins belonging to the 58 families and suggest hypotheses about mechanism for the subset for which the function is known, and hypotheses about function for the remainder. }, keywords = {}, pubstate = {published}, tppubtype = {article} } The prediction of the structures of proteins without detectable sequence similarity to any protein of known structure remains an outstanding scientific challenge. Here we report significant progress in this area. We first describe de novo blind structure predictions of unprecendented accuracy we made for two proteins in large families in the recent CASP11 blind test of protein structure prediction methods by incorporating residue-residue co-evolution information in the Rosetta structure prediction program. We then describe the use of this method to generate structure models for 58 of the 121 large protein families in prokaryotes for which three-dimensional structures are not available. These models, which are posted online for public access, provide structural information for the over 400,000 proteins belonging to the 58 families and suggest hypotheses about mechanism for the subset for which the function is known, and hypotheses about function for the remainder. |
Wolf, Clancey; Siegel, Justin B; Tinberg, Christine; Camarca, Alessandra; Gianfrani, Carmen; Paski, Shirley; Guan, Rongjin; Montelione, Gaetano T; Baker, David; Pultz, Ingrid S Engineering of Kuma030: a gliadin peptidase that rapidly degrades immunogenic gliadin peptides in gastric conditions. Journal Article Journal of the American Chemical Society, 2015, ISSN: 1520-5126. @article{617, title = {Engineering of Kuma030: a gliadin peptidase that rapidly degrades immunogenic gliadin peptides in gastric conditions.}, author = { Clancey Wolf and Justin B Siegel and Christine Tinberg and Alessandra Camarca and Carmen Gianfrani and Shirley Paski and Rongjin Guan and Gaetano T Montelione and David Baker and Ingrid S Pultz}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Wolf_JACS_2015.pdf}, doi = {10.1021/jacs.5b08325}, issn = {1520-5126}, year = {2015}, date = {2015-09-01}, journal = {Journal of the American Chemical Society}, abstract = {Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions with minutes, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions with minutes, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease. |
Bale, Jacob B; Park, Rachel U; Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Cascio, Duilio; King, Neil P; Yeates, Todd O; Baker, David Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression Journal Article Protein science : a publication of the Protein Society, 2015, ISSN: 1469-896X. @article{616, title = {Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression}, author = { Jacob B Bale and Rachel U Park and Yuxi Liu and Shane Gonen and Tamir Gonen and Duilio Cascio and Neil P. King and Todd O. Yeates and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Bale_designed_tetrahedral_ProteinSci2015.pdf}, doi = {10.1002/pro.2748}, issn = {1469-896X}, year = {2015}, date = {2015-07-01}, journal = {Protein science : a publication of the Protein Society}, abstract = {We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials. |
Gonen, Shane; DiMaio, Frank; Gonen, Tamir; Baker, David Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces Journal Article Science (New York, N.Y.), 348 , pp. 1365-8, 2015, ISSN: 1095-9203. @article{613, title = {Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces}, author = { Shane Gonen and Frank DiMaio and Tamir Gonen and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Gonen_2DArrays_Baker2015.pdf}, doi = {10.1126/science.aaa9897}, issn = {1095-9203}, year = {2015}, date = {2015-06-01}, journal = {Science (New York, N.Y.)}, volume = {348}, pages = {1365-8}, abstract = {We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering. |
Park, Hahnbeom; DiMaio, Frank; Baker, David The origin of consistent protein structure refinement from structural averaging. Journal Article Structure (London, England : 1993), 23 , pp. 1123-8, 2015, ISSN: 1878-4186. @article{615, title = {The origin of consistent protein structure refinement from structural averaging.}, author = { Hahnbeom Park and Frank DiMaio and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Park_Structure_2015.pdf http://www.ncbi.nlm.nih.gov/pubmed/?term=The+Origin+of+Consistent+Protein+Structure+Refinement+from+Structural+Averaging}, doi = {10.1016/j.str.2015.03.022}, issn = {1878-4186}, year = {2015}, date = {2015-06-01}, journal = {Structure (London, England : 1993)}, volume = {23}, pages = {1123-8}, abstract = {Recent studies have shown that explicit solvent molecular dynamics (MD) simulation followed by structural averaging can consistently improve protein structure models. We find that improvement upon averaging is not limited to explicit water MD simulation, as consistent improvements are also observed for more efficient implicit solvent MD or Monte Carlo minimization simulations. To determine the origin of these improvements, we examine the changes in model accuracy brought about by averaging at the individual residue level. We find that the improvement in model quality from averaging results from the superposition of two effects: a dampening of deviations from the correct structure in the least well modeled regions, and a reinforcement of consistent movements towards the correct structure in better modeled regions. These observations are consistent with an energy landscape model in which the magnitude of the energy gradient toward the native structure decreases with increasing distance from the native state.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Recent studies have shown that explicit solvent molecular dynamics (MD) simulation followed by structural averaging can consistently improve protein structure models. We find that improvement upon averaging is not limited to explicit water MD simulation, as consistent improvements are also observed for more efficient implicit solvent MD or Monte Carlo minimization simulations. To determine the origin of these improvements, we examine the changes in model accuracy brought about by averaging at the individual residue level. We find that the improvement in model quality from averaging results from the superposition of two effects: a dampening of deviations from the correct structure in the least well modeled regions, and a reinforcement of consistent movements towards the correct structure in better modeled regions. These observations are consistent with an energy landscape model in which the magnitude of the energy gradient toward the native structure decreases with increasing distance from the native state. |
Siegel, Justin B; Smith, Amanda Lee; Poust, Sean; Wargacki, Adam J; Bar-Even, Arren; Louw, Catherine; Shen, Betty W; Eiben, Christopher B; Tran, Huu M; Noor, Elad; Gallaher, Jasmine L; Bale, Jacob; Yoshikuni, Yasuo; Gelb, Michael H; Keasling, Jay D; Stoddard, Barry L; Lidstrom, Mary E; Baker, David Computational protein design enables a novel one-carbon assimilation pathway Journal Article Proceedings of the National Academy of Sciences of the United States of America, 2015, ISSN: 1091-6490. @article{565, title = {Computational protein design enables a novel one-carbon assimilation pathway}, author = { Justin B Siegel and Amanda Lee Smith and Sean Poust and Adam J Wargacki and Arren Bar-Even and Catherine Louw and Betty W Shen and Christopher B Eiben and Huu M Tran and Elad Noor and Jasmine L Gallaher and Jacob Bale and Yasuo Yoshikuni and Michael H Gelb and Jay D Keasling and Barry L Stoddard and Mary E Lidstrom and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/siegel15A.pdf}, doi = {10.1073/pnas.1500545112}, issn = {1091-6490}, year = {2015}, date = {2015-03-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, abstract = {We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway. |
DiMaio, Frank; Song, Yifan; Li, Xueming; Brunner, Matthias J; Xu, Chunfu; Conticello, Vincent; Egelman, Edward; Marlovits, Thomas C; Cheng, Yifan; Baker, David Atomic-accuracy models from 4.5-r A cryo-electron microscopy data with density-guided iterative local refinement. Journal Article Nature methods, 2015, ISSN: 1548-7105. @article{560, title = {Atomic-accuracy models from 4.5-r A cryo-electron microscopy data with density-guided iterative local refinement.}, author = { Frank DiMaio and Yifan Song and Xueming Li and Matthias J Brunner and Chunfu Xu and Vincent Conticello and Edward Egelman and Thomas C Marlovits and Yifan Cheng and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/DiMaio_NatMethods_2015.pdf}, doi = {10.1038/nmeth.3286}, issn = {1548-7105}, year = {2015}, date = {2015-02-01}, journal = {Nature methods}, abstract = {We describe a general approach for refining protein structure models on the basis of cryo-electron microscopy maps with near-atomic resolution. The method integrates Monte Carlo sampling with local density-guided optimization, Rosetta all-atom refinement and real-space B-factor fitting. In tests on experimental maps of three different systems with 4.5-r A resolution or better, the method consistently produced models with atomic-level accuracy largely independently of starting-model quality, and it outperformed the molecular dynamics-based MDFF method. Cross-validated model quality statistics correlated with model accuracy over the three test systems.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a general approach for refining protein structure models on the basis of cryo-electron microscopy maps with near-atomic resolution. The method integrates Monte Carlo sampling with local density-guided optimization, Rosetta all-atom refinement and real-space B-factor fitting. In tests on experimental maps of three different systems with 4.5-r A resolution or better, the method consistently produced models with atomic-level accuracy largely independently of starting-model quality, and it outperformed the molecular dynamics-based MDFF method. Cross-validated model quality statistics correlated with model accuracy over the three test systems. |
Wang, Ray Yu-Ruei; Kudryashev, Mikhail; Li, Xueming; Egelman, Edward H; Basler, Marek; Cheng, Yifan; Baker, David; DiMaio, Frank De novo protein structure determination from near-atomic-resolution cryo-EM maps. Journal Article Nature methods, 2015, ISSN: 1548-7105. @article{559, title = {De novo protein structure determination from near-atomic-resolution cryo-EM maps.}, author = { Ray Yu-Ruei Wang and Mikhail Kudryashev and Xueming Li and Edward H Egelman and Marek Basler and Yifan Cheng and David Baker and Frank DiMaio}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Wang_NatMethods_2015.pdf}, doi = {10.1038/nmeth.3287}, issn = {1548-7105}, year = {2015}, date = {2015-02-01}, journal = {Nature methods}, abstract = {We present a de novo model-building approach that combines predicted backbone conformations with side-chain fit to density to accurately assign sequence into density maps. This method yielded accurate models for six of nine experimental maps at 3.3- to 4.8-r A resolution and produced a nearly complete model for an unsolved map containing a 660-residue heterodimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic-resolution cryo-electron microscopy (cryo-EM) maps.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We present a de novo model-building approach that combines predicted backbone conformations with side-chain fit to density to accurately assign sequence into density maps. This method yielded accurate models for six of nine experimental maps at 3.3- to 4.8-r A resolution and produced a nearly complete model for an unsolved map containing a 660-residue heterodimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic-resolution cryo-electron microscopy (cryo-EM) maps. |
Rossi, Paolo; Shi, Lei; Liu, Gaohua; Barbieri, Christopher M; Lee, Hsiau-Wei; Grant, Thomas D; Luft, Joseph R; Xiao, Rong; Acton, Thomas B; Snell, Edward H; Montelione, Gaetano T; Baker, David; Lange, Oliver F; Sgourakis, Nikolaos G A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta Journal Article Proteins, 83 , pp. 309-17, 2015, ISSN: 1097-0134. @article{611, title = {A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta}, author = { Paolo Rossi and Lei Shi and Gaohua Liu and Christopher M Barbieri and Hsiau-Wei Lee and Thomas D Grant and Joseph R Luft and Rong Xiao and Thomas B Acton and Edward H Snell and Gaetano T Montelione and David Baker and Oliver F Lange and Nikolaos G Sgourakis}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/ahybridnmrsaxsbased_Baker2015.pdf}, doi = {10.1002/prot.24719}, issn = {1097-0134}, year = {2015}, date = {2015-02-01}, journal = {Proteins}, volume = {83}, pages = {309-17}, abstract = {Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution. |
Pearson, Aaron D; Mills, Jeremy H; Song, Yifan; Nasertorabi, Fariborz; Han, Gye Won; Baker, David; Stevens, Raymond C; Schultz, Peter G Transition states. Trapping a transition state in a computationally designed protein bottle. Journal Article Science (New York, N.Y.), 347 , pp. 863-7, 2015, ISSN: 1095-9203. @article{561, title = {Transition states. Trapping a transition state in a computationally designed protein bottle.}, author = { Aaron D Pearson and Jeremy H Mills and Yifan Song and Fariborz Nasertorabi and Gye Won Han and David Baker and Raymond C Stevens and Peter G Schultz}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Mills_Science_2015A.pdf}, doi = {10.1126/science.aaa2424}, issn = {1095-9203}, year = {2015}, date = {2015-02-01}, journal = {Science (New York, N.Y.)}, volume = {347}, pages = {863-7}, abstract = {The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Here, we used packing interactions within the core of a protein to stabilize the planar TS conformation for rotation around the central carbon-carbon bond of biphenyl so that it could be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis, we identified a protein in which the side chain of p-biphenylalanine is trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Here, we used packing interactions within the core of a protein to stabilize the planar TS conformation for rotation around the central carbon-carbon bond of biphenyl so that it could be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis, we identified a protein in which the side chain of p-biphenylalanine is trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction. |
Egelman, E H; Xu, C; DiMaio, F; Magnotti, E; Modlin, C; Yu, X; Wright, E; Baker, D; Conticello, V P Structural plasticity of helical nanotubes based on coiled-coil assemblies. Journal Article Structure (London, England : 1993), 23 , pp. 280-9, 2015, ISSN: 1878-4186. @article{609, title = {Structural plasticity of helical nanotubes based on coiled-coil assemblies.}, author = { E H Egelman and C. Xu and F. DiMaio and E Magnotti and C Modlin and X Yu and E Wright and D Baker and V P Conticello}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/structuralplasticity_Baker2015.pdf}, doi = {10.1016/j.str.2014.12.008}, issn = {1878-4186}, year = {2015}, date = {2015-02-01}, journal = {Structure (London, England : 1993)}, volume = {23}, pages = {280-9}, abstract = {Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies. |
Morag, Omry; Sgourakis, Nikolaos G; Baker, David; Goldbourt, Amir The NMR-Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope Journal Article Proceedings of the National Academy of Sciences of the United States of America, 112 , pp. 971-6, 2015, ISSN: 1091-6490. @article{607, title = {The NMR-Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope}, author = { Omry Morag and Nikolaos G Sgourakis and David Baker and Amir Goldbourt}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/thenmrrosettacapsid_Baker2015.pdf}, doi = {10.1073/pnas.1415393112}, issn = {1091-6490}, year = {2015}, date = {2015-01-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, pages = {971-6}, abstract = {Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ~1 μm and a diameter of ~7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6-16.7 r A and a tilt of 36.1-36.6textdegree between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ~1 μm and a diameter of ~7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6-16.7 r A and a tilt of 36.1-36.6textdegree between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies. |
Vittal, Vinayak; Shi, Lei; Wenzel, Dawn M; Scaglione, Matthew K; Duncan, Emily D; Basrur, Venkatesha; Elenitoba-Johnson, Kojo S J; Baker, David; Paulson, Henry L; Brzovic, Peter S; Klevit, Rachel E Intrinsic disorder drives N-terminal ubiquitination by Ube2w Journal Article Nature Chemical Biology, 11 , pp. 83-9, 2015, ISSN: 1552-4469. @article{610, title = {Intrinsic disorder drives N-terminal ubiquitination by Ube2w}, author = { Vinayak Vittal and Lei Shi and Dawn M Wenzel and K Matthew Scaglione and Emily D Duncan and Venkatesha Basrur and Kojo S J Elenitoba-Johnson and David Baker and Henry L Paulson and Peter S Brzovic and Rachel E Klevit}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/intrinsicdisorderdrives_Baker2015.pdf}, doi = {10.1038/nchembio.1700}, issn = {1552-4469}, year = {2015}, date = {2015-01-01}, journal = {Nature Chemical Biology}, volume = {11}, pages = {83-9}, abstract = {Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells. |
Park, Keunwan; Shen, Betty W; Parmeggiani, Fabio; Huang, Po-Ssu; Stoddard, Barry L; Baker, David Control of repeat-protein curvature by computational protein design. Journal Article Nature structural & molecular biology, 2015, ISSN: 1545-9985. @article{557, title = {Control of repeat-protein curvature by computational protein design.}, author = { Keunwan Park and Betty W Shen and Fabio Parmeggiani and Po-Ssu Huang and Barry L Stoddard and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Park_2015.pdf}, doi = {10.1038/nsmb.2938}, issn = {1545-9985}, year = {2015}, date = {2015-01-01}, journal = {Nature structural & molecular biology}, abstract = {Shape complementarity is an important component of molecular recognition, and the ability to precisely adjust the shape of a binding scaffold to match a target of interest would greatly facilitate the creation of high-affinity protein reagents and therapeutics. Here we describe a general approach to control the shape of the binding surface on repeat-protein scaffolds and apply it to leucine-rich-repeat proteins. First, self-compatible building-block modules are designed that, when polymerized, generate surfaces with unique but constant curvatures. Second, a set of junction modules that connect the different building blocks are designed. Finally, new proteins with custom-designed shapes are generated by appropriately combining building-block and junction modules. Crystal structures of the designs illustrate the power of the approach in controlling repeat-protein curvature.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Shape complementarity is an important component of molecular recognition, and the ability to precisely adjust the shape of a binding scaffold to match a target of interest would greatly facilitate the creation of high-affinity protein reagents and therapeutics. Here we describe a general approach to control the shape of the binding surface on repeat-protein scaffolds and apply it to leucine-rich-repeat proteins. First, self-compatible building-block modules are designed that, when polymerized, generate surfaces with unique but constant curvatures. Second, a set of junction modules that connect the different building blocks are designed. Finally, new proteins with custom-designed shapes are generated by appropriately combining building-block and junction modules. Crystal structures of the designs illustrate the power of the approach in controlling repeat-protein curvature. |
Bergeron, Julien R C; Worrall, Liam J; De, Soumya; Sgourakis, Nikolaos G; Cheung, Adrienne H; Lameignere, Emilie; Okon, Mark; Wasney, Gregory A; Baker, David; McIntosh, Lawrence P; Strynadka, Natalie C J The modular structure of the inner-membrane ring component PrgK facilitates assembly of the type III secretion system basal body Journal Article Structure (London, England : 1993), 23 , pp. 161-72, 2015, ISSN: 1878-4186. @article{608, title = {The modular structure of the inner-membrane ring component PrgK facilitates assembly of the type III secretion system basal body}, author = { Julien R C Bergeron and Liam J Worrall and Soumya De and Nikolaos G Sgourakis and Adrienne H Cheung and Emilie Lameignere and Mark Okon and Gregory A Wasney and David Baker and Lawrence P McIntosh and Natalie C J Strynadka}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/themodularstructure_Baker2015.pdf}, doi = {10.1016/j.str.2014.10.021}, issn = {1878-4186}, year = {2015}, date = {2015-01-01}, journal = {Structure (London, England : 1993)}, volume = {23}, pages = {161-72}, abstract = {The type III secretion system (T3SS) is a large macromolecular assembly found at the surface of many pathogenic Gram-negative bacteria. Its role is to inject toxic "effector" proteins into the cells of infected organisms. The molecular details of the assembly of this large, multimembrane-spanning complex remain poorly understood. Here, we report structural, biochemical, and functional analyses of PrgK, an inner-membrane component of the prototypical Salmonella typhimurium T3SS. We have obtained the atomic structures of the two ring building globular domains and show that the C-terminal transmembrane helix is not essential for assembly and secretion. We also demonstrate that structural rearrangement of the two PrgK globular domains, driven by an interconnecting linker region, may promote oligomerization into ring structures. Finally, we used electron microscopy-guided symmetry modeling to propose a structural model for the intimately associated PrgH-PrgK ring interaction within the assembled basal body.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The type III secretion system (T3SS) is a large macromolecular assembly found at the surface of many pathogenic Gram-negative bacteria. Its role is to inject toxic "effector" proteins into the cells of infected organisms. The molecular details of the assembly of this large, multimembrane-spanning complex remain poorly understood. Here, we report structural, biochemical, and functional analyses of PrgK, an inner-membrane component of the prototypical Salmonella typhimurium T3SS. We have obtained the atomic structures of the two ring building globular domains and show that the C-terminal transmembrane helix is not essential for assembly and secretion. We also demonstrate that structural rearrangement of the two PrgK globular domains, driven by an interconnecting linker region, may promote oligomerization into ring structures. Finally, we used electron microscopy-guided symmetry modeling to propose a structural model for the intimately associated PrgH-PrgK ring interaction within the assembled basal body. |
2014 |
Thyme, Summer B; Song, Yifan; Brunette, Tj; Szeto, Mindy D; Kusak, Lara; Bradley, Philip; Baker, David Massively parallel determination and modeling of endonuclease substrate specificity Journal Article Nucleic Acids Research, 42 , pp. 13839-52, 2014, ISSN: 1362-4962. @article{606, title = {Massively parallel determination and modeling of endonuclease substrate specificity}, author = { Summer B Thyme and Yifan Song and Tj Brunette and Mindy D Szeto and Lara Kusak and Philip Bradley and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/massivelyparallel_Baker2013.pdf}, doi = {10.1093/nar/gku1096}, issn = {1362-4962}, year = {2014}, date = {2014-12-01}, journal = {Nucleic Acids Research}, volume = {42}, pages = {13839-52}, abstract = {We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution. |
Parmeggiani, Fabio; Huang, Po-Ssu; Vorobiev, Sergey; Xiao, Rong; Park, Keunwan; Caprari, Silvia; Su, Min; Seetharaman, Jayaraman; Mao, Lei; Janjua, Haleema; Montelione, Gaetano T; Hunt, John; Baker, David A General Computational Approach for Repeat Protein Design. Journal Article Journal of molecular biology, 2014, ISSN: 1089-8638. @article{555, title = {A General Computational Approach for Repeat Protein Design.}, author = { Fabio Parmeggiani and Po-Ssu Huang and Sergey Vorobiev and Rong Xiao and Keunwan Park and Silvia Caprari and Min Su and Jayaraman Seetharaman and Lei Mao and Haleema Janjua and Gaetano T Montelione and John Hunt and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Parmeggiani-2014.pdf}, doi = {10.1016/j.jmb.2014.11.005}, issn = {1089-8638}, year = {2014}, date = {2014-11-01}, journal = {Journal of molecular biology}, abstract = {Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1r A root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1r A root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds. |
Liu, Daniel S; Niv'on, Lucas G; Richter, Florian; Goldman, Peter J; Deerinck, Thomas J; Yao, Jennifer Z; Richardson, Douglas; Phipps, William S; Ye, Anne Z; Ellisman, Mark H; Drennan, Catherine L; Baker, David; Ting, Alice Y Computational design of a red fluorophore ligase for site-specific protein labeling in living cells. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 111 , pp. E4551-9, 2014, ISSN: 1091-6490. @article{619, title = {Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.}, author = { Daniel S Liu and Lucas G Niv'on and Florian Richter and Peter J Goldman and Thomas J Deerinck and Jennifer Z Yao and Douglas Richardson and William S Phipps and Anne Z Ye and Mark H Ellisman and Catherine L Drennan and David Baker and Alice Y Ting}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Liu_computational_PNAS_2014.pdf}, doi = {10.1073/pnas.1404736111}, issn = {1091-6490}, year = {2014}, date = {2014-10-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, pages = {E4551-9}, abstract = {Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies. |
Huang, P; Oberdorfer, G; Xu, C; Pei, X Y; Nannenga, B L; Rogers, J M; DiMaio, F; Gonen, T; Luisi, B; Baker, D High thermodynamic stability of parametrically designed helical bundles Journal Article Science, 346 , pp. 481-85, 2014. @article{552, title = {High thermodynamic stability of parametrically designed helical bundles}, author = { P. Huang and G. Oberdorfer and C. Xu and X.Y. Pei and B.L. Nannenga and J.M. Rogers and F. DiMaio and T. Gonen and B. Luisi and D Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Huang_2014.pdf}, doi = {10.1126/science.1257481}, year = {2014}, date = {2014-10-01}, journal = {Science}, volume = {346}, pages = {481-85}, chapter = {481}, abstract = {We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil–generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil–generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications. |
Liu, Yu; Zhang, Xin; Tan, Yun Lei; Bhabha, Gira; Ekiert, Damian C; Kipnis, Yakov; Bjelic, Sinisa; Baker, David; Kelly, Jeffery W De novo-designed enzymes as small-molecule-regulated fluorescence imaging tags and fluorescent reporters. Journal Article Journal of the American Chemical Society, 136 , pp. 13102-5, 2014, ISSN: 1520-5126. @article{621, title = {De novo-designed enzymes as small-molecule-regulated fluorescence imaging tags and fluorescent reporters.}, author = { Yu Liu and Xin Zhang and Yun Lei Tan and Gira Bhabha and Damian C Ekiert and Yakov Kipnis and Sinisa Bjelic and David Baker and Jeffery W Kelly}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Liu_JACS_2014.pdf}, doi = {10.1021/ja5056356}, issn = {1520-5126}, year = {2014}, date = {2014-09-01}, journal = {Journal of the American Chemical Society}, volume = {136}, pages = {13102-5}, abstract = {Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme-small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme-small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores. |
Khoury, George A; Liwo, Adam; Khatib, Firas; Zhou, Hongyi; Chopra, Gaurav; Bacardit, Jaume; Bortot, Leandro O; Faccioli, Rodrigo A; Deng, Xin; He, Yi; Krupa, Pawel; Li, Jilong; Mozolewska, Magdalena A; Sieradzan, Adam K; Smadbeck, James; Wirecki, Tomasz; Cooper, Seth; Flatten, Jeff; Xu, Kefan; Baker, David; Cheng, Jianlin; Delbem, Alexandre C B; Floudas, Christodoulos A; Keasar, Chen; Levitt, Michael; Popovi'c, Zoran; Scheraga, Harold A; Skolnick, Jeffrey; Crivelli, Silvia N WeFold: a coopetition for protein structure prediction. Journal Article Proteins, 82 , pp. 1850-68, 2014, ISSN: 1097-0134. @article{625, title = {WeFold: a coopetition for protein structure prediction.}, author = { George A Khoury and Adam Liwo and Firas Khatib and Hongyi Zhou and Gaurav Chopra and Jaume Bacardit and Leandro O Bortot and Rodrigo A Faccioli and Xin Deng and Yi He and Pawel Krupa and Jilong Li and Magdalena A Mozolewska and Adam K Sieradzan and James Smadbeck and Tomasz Wirecki and Seth Cooper and Jeff Flatten and Kefan Xu and David Baker and Jianlin Cheng and Alexandre C B Delbem and Christodoulos A Floudas and Chen Keasar and Michael Levitt and Zoran Popovi'c and Harold A Scheraga and Jeffrey Skolnick and Silvia N Crivelli}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Khoury_Proteins_2014.pdf}, doi = {10.1002/prot.24538}, issn = {1097-0134}, year = {2014}, date = {2014-09-01}, journal = {Proteins}, volume = {82}, pages = {1850-68}, abstract = {The protein structure prediction problem continues to elude scientists. Despite the introduction of many methods, only modest gains were made over the last decade for certain classes of prediction targets. To address this challenge, a social-media based worldwide collaborative effort, named WeFold, was undertaken by 13 labs. During the collaboration, the laboratories were simultaneously competing with each other. Here, we present the first attempt at "coopetition" in scientific research applied to the protein structure prediction and refinement problems. The coopetition was possible by allowing the participating labs to contribute different components of their protein structure prediction pipelines and create new hybrid pipelines that they tested during CASP10. This manuscript describes both successes and areas needing improvement as identified throughout the first WeFold experiment and discusses the efforts that are underway to advance this initiative. A footprint of all contributions and structures are publicly accessible at http://www.wefold.org.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The protein structure prediction problem continues to elude scientists. Despite the introduction of many methods, only modest gains were made over the last decade for certain classes of prediction targets. To address this challenge, a social-media based worldwide collaborative effort, named WeFold, was undertaken by 13 labs. During the collaboration, the laboratories were simultaneously competing with each other. Here, we present the first attempt at "coopetition" in scientific research applied to the protein structure prediction and refinement problems. The coopetition was possible by allowing the participating labs to contribute different components of their protein structure prediction pipelines and create new hybrid pipelines that they tested during CASP10. This manuscript describes both successes and areas needing improvement as identified throughout the first WeFold experiment and discusses the efforts that are underway to advance this initiative. A footprint of all contributions and structures are publicly accessible at http://www.wefold.org. |
Griss, Rudolf; Schena, Alberto; Reymond, Luc; Patiny, Luc; Werner, Dominique; Tinberg, Christine E; Baker, David; Johnsson, Kai Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring Journal Article Nature chemical biology, 10 , pp. 598-603, 2014, ISSN: 1552-4469. @article{539, title = {Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring}, author = { Rudolf Griss and Alberto Schena and Luc Reymond and Luc Patiny and Dominique Werner and Christine E Tinberg and David Baker and Kai Johnsson}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Griss_2014A.pdf}, doi = {10.1038/nchembio.1554}, issn = {1552-4469}, year = {2014}, date = {2014-07-01}, journal = {Nature chemical biology}, volume = {10}, pages = {598-603}, abstract = {For many drugs, finding the balance between efficacy and toxicity requires monitoring their concentrations in the patienttextquoterights blood. Quantifying drug levels at the bedside or at home would have advantages in terms of therapeutic outcome and convenience, but current techniques require the setting of a diagnostic laboratory. We have developed semisynthetic bioluminescent sensors that permit precise measurements of drug concentrations in patient samples by spotting minimal volumes on paper and recording the signal using a simple point-and-shoot camera. Our sensors have a modular design consisting of a protein-based and a synthetic part and can be engineered to selectively recognize a wide range of drugs, including immunosuppressants, antiepileptics, anticancer agents and antiarrhythmics. This low-cost point-of-care method could make therapies safer, increase the convenience of doctors and patients and make therapeutic drug monitoring available in regions with poor infrastructure.}, keywords = {}, pubstate = {published}, tppubtype = {article} } For many drugs, finding the balance between efficacy and toxicity requires monitoring their concentrations in the patienttextquoterights blood. Quantifying drug levels at the bedside or at home would have advantages in terms of therapeutic outcome and convenience, but current techniques require the setting of a diagnostic laboratory. We have developed semisynthetic bioluminescent sensors that permit precise measurements of drug concentrations in patient samples by spotting minimal volumes on paper and recording the signal using a simple point-and-shoot camera. Our sensors have a modular design consisting of a protein-based and a synthetic part and can be engineered to selectively recognize a wide range of drugs, including immunosuppressants, antiepileptics, anticancer agents and antiarrhythmics. This low-cost point-of-care method could make therapies safer, increase the convenience of doctors and patients and make therapeutic drug monitoring available in regions with poor infrastructure. |