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2016 |
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. |
Preiswerk, Nathalie; Beck, Tobias; Schulz, Jessica D; Milovn'ik, Peter; Mayer, Clemens; Siegel, Justin B; Baker, David; Hilvert, Donald Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 111 , pp. 8013-8, 2014, ISSN: 1091-6490. @article{623, title = {Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase.}, author = { Nathalie Preiswerk and Tobias Beck and Jessica D Schulz and Peter Milovn'ik and Clemens Mayer and Justin B Siegel and David Baker and Donald Hilvert}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Preiswerk_PNAS_2014.pdf}, doi = {10.1073/pnas.1401073111}, issn = {1091-6490}, year = {2014}, date = {2014-06-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, pages = {8013-8}, abstract = {By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.}, keywords = {}, pubstate = {published}, tppubtype = {article} } By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions. |
Mazor, Ronit; Eberle, Jaime A; Hu, Xiaobo; Vassall, Aaron N; Onda, Masanori; Beers, Richard; Lee, Elizabeth C; Kreitman, Robert J; Lee, Byungkook; Baker, David; King, Chris; Hassan, Raffit; Benhar, Itai; Pastan, Ira Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 111 , pp. 8571-6, 2014, ISSN: 1091-6490. @article{624, title = {Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes.}, author = { Ronit Mazor and Jaime A Eberle and Xiaobo Hu and Aaron N Vassall and Masanori Onda and Richard Beers and Elizabeth C Lee and Robert J Kreitman and Byungkook Lee and David Baker and Chris King and Raffit Hassan and Itai Benhar and Ira Pastan}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Mazor_PNAS_2014.pdf}, doi = {10.1073/pnas.1405153111}, issn = {1091-6490}, year = {2014}, date = {2014-06-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, pages = {8571-6}, abstract = {Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins. |
Magad'an, Javier G; Altman, Meghan O; Ince, William L; Hickman, Heather D; Stevens, James; Chevalier, Aaron; Baker, David; Wilson, Patrick C; Ahmed, Rafi; Bennink, Jack R; Yewdell, Jonathan W Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes. Journal Article PLoS pathogens, 10 , pp. e1004204, 2014, ISSN: 1553-7374. @article{537, title = {Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes.}, author = { Javier G Magad'an and Meghan O Altman and William L Ince and Heather D Hickman and James Stevens and Aaron Chevalier and David Baker and Patrick C Wilson and Rafi Ahmed and Jack R Bennink and Jonathan W Yewdell}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Magadan_2014A.pdf}, doi = {10.1371/journal.ppat.1004204}, issn = {1553-7374}, year = {2014}, date = {2014-06-01}, journal = {PLoS pathogens}, volume = {10}, pages = {e1004204}, abstract = {Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4textdegreeC and well at 37textdegreeC. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4textdegreeC and well at 37textdegreeC. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans. |
Procko, Erik; Berguig, Geoffrey Y; Shen, Betty W; Song, Yifan; Frayo, Shani; Convertine, Anthony J; Margineantu, Daciana; Booth, Garrett; Correia, Bruno E; Cheng, Yuanhua; Schief, William R; Hockenbery, David M; Press, Oliver W; Stoddard, Barry L; Stayton, Patrick S; Baker, David A computationally designed inhibitor of an epstein-barr viral bcl-2 protein induces apoptosis in infected cells. Journal Article Cell, 157 , pp. 1644-56, 2014, ISSN: 1097-4172. @article{538, title = {A computationally designed inhibitor of an epstein-barr viral bcl-2 protein induces apoptosis in infected cells.}, author = { Erik Procko and Geoffrey Y Berguig and Betty W Shen and Yifan Song and Shani Frayo and Anthony J Convertine and Daciana Margineantu and Garrett Booth and Bruno E Correia and Yuanhua Cheng and William R Schief and David M Hockenbery and Oliver W Press and Barry L Stoddard and Patrick S Stayton and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Procko2014A.pdf}, doi = {10.1016/j.cell.2014.04.034}, issn = {1097-4172}, year = {2014}, date = {2014-06-01}, journal = {Cell}, volume = {157}, pages = {1644-56}, abstract = {Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates. |
Chen, Kuang-Yui M; Sun, Jiaming; Salvo, Jason S; Baker, David; Barth, Patrick High-resolution modeling of transmembrane helical protein structures from distant homologues. Journal Article PLoS computational biology, 10 , pp. e1003636, 2014, ISSN: 1553-7358. @article{622, title = {High-resolution modeling of transmembrane helical protein structures from distant homologues.}, author = { Kuang-Yui M Chen and Jiaming Sun and Jason S Salvo and David Baker and Patrick Barth}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Chen_PLOS_2014.pdf}, doi = {10.1371/journal.pcbi.1003636}, issn = {1553-7358}, year = {2014}, date = {2014-05-01}, journal = {PLoS computational biology}, volume = {10}, pages = {e1003636}, abstract = {Eukaryotic transmembrane helical (TMH) proteins perform a wide diversity of critical cellular functions, but remain structurally largely uncharacterized and their high-resolution structure prediction is currently hindered by the lack of close structural homologues. To address this problem, we present a novel and generic method for accurately modeling large TMH protein structures from distant homologues exhibiting distinct loop and TMH conformations. Models of the adenosine A2AR and chemokine CXCR4 receptors were first ranked in GPCR-DOCK blind prediction contests in the receptor structure accuracy category. In a benchmark of 50 TMH protein homolog pairs of diverse topology (from 5 to 12 TMHs), size (from 183 to 420 residues) and sequence identity (from 15% to 70%), the method improves most starting templates, and achieves near-atomic accuracy prediction of membrane-embedded regions. Unlike starting templates, the models are of suitable quality for computer-based protein engineering: redesigned models and redesigned X-ray structures exhibit very similar native interactions. The method should prove useful for the atom-level modeling and design of a large fraction of structurally uncharacterized TMH proteins from a wide range of structural homologues.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Eukaryotic transmembrane helical (TMH) proteins perform a wide diversity of critical cellular functions, but remain structurally largely uncharacterized and their high-resolution structure prediction is currently hindered by the lack of close structural homologues. To address this problem, we present a novel and generic method for accurately modeling large TMH protein structures from distant homologues exhibiting distinct loop and TMH conformations. Models of the adenosine A2AR and chemokine CXCR4 receptors were first ranked in GPCR-DOCK blind prediction contests in the receptor structure accuracy category. In a benchmark of 50 TMH protein homolog pairs of diverse topology (from 5 to 12 TMHs), size (from 183 to 420 residues) and sequence identity (from 15% to 70%), the method improves most starting templates, and achieves near-atomic accuracy prediction of membrane-embedded regions. Unlike starting templates, the models are of suitable quality for computer-based protein engineering: redesigned models and redesigned X-ray structures exhibit very similar native interactions. The method should prove useful for the atom-level modeling and design of a large fraction of structurally uncharacterized TMH proteins from a wide range of structural homologues. |
King, Neil P; Bale, Jacob B; Sheffler, William; McNamara, Dan E; Gonen, Shane; Gonen, Tamir; Yeates, Todd O; Baker, David Accurate design of co-assembling multi-component protein nanomaterials. Journal Article Nature, 2014, ISSN: 1476-4687. @article{534, title = {Accurate design of co-assembling multi-component protein nanomaterials.}, author = { Neil P. King and Jacob B Bale and William Sheffler and Dan E McNamara and Shane Gonen and Tamir Gonen and Todd O. Yeates and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/King_Nature2014A.pdf}, doi = {10.1038/nature13404}, issn = {1476-4687}, year = {2014}, date = {2014-05-01}, journal = {Nature}, abstract = {The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications. |
Alushin, Gregory M; Lander, Gabriel C; Kellogg, Elizabeth H; Zhang, Rui; Baker, David; Nogales, Eva High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Journal Article Cell, 157 , pp. 1117-29, 2014, ISSN: 1097-4172. @article{541, title = {High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis.}, author = { Gregory M Alushin and Gabriel C Lander and Elizabeth H Kellogg and Rui Zhang and David Baker and Eva Nogales}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Alushin_2014A.pdf}, doi = {10.1016/j.cell.2014.03.053}, issn = {1097-4172}, year = {2014}, date = {2014-05-01}, journal = {Cell}, volume = {157}, pages = {1117-29}, abstract = {Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 r A) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 r A) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces. |
King, Chris; Garza, Esteban N; Mazor, Ronit; Linehan, Jonathan L; Pastan, Ira; Pepper, Marion; Baker, David Removing T-cell epitopes with computational protein design. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 2014, ISSN: 1091-6490. @article{533, title = {Removing T-cell epitopes with computational protein design.}, author = { Chris King and Esteban N Garza and Ronit Mazor and Jonathan L Linehan and Ira Pastan and Marion Pepper and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/King_PNAS_2014A.pdf}, doi = {10.1073/pnas.1321126111}, issn = {1091-6490}, year = {2014}, date = {2014-05-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, abstract = {Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function. |
Ovchinnikov, Sergey; Kamisetty, Hetunandan; Baker, David Robust and accurate prediction of residue-residue interactions across protein interfaces using evolutionary information. Journal Article eLife, 3 , pp. e02030, 2014, ISSN: 2050-084X. @article{540, title = {Robust and accurate prediction of residue-residue interactions across protein interfaces using evolutionary information.}, author = { Sergey Ovchinnikov and Hetunandan Kamisetty and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Ovchinnikov_2014A.pdf}, doi = {10.7554/eLife.02030}, issn = {2050-084X}, year = {2014}, date = {2014-05-01}, journal = {eLife}, volume = {3}, pages = {e02030}, abstract = {Do the amino acid sequence identities of residues that make contact across protein interfaces covary during evolution? If so, such covariance could be used to predict contacts across interfaces and assemble models of biological complexes. We find that residue pairs identified using a pseudo-likelihood-based method to covary across protein-protein interfaces in the 50S ribosomal unit and 28 additional bacterial protein complexes with known structure are almost always in contact in the complex, provided that the number of aligned sequences is greater than the average length of the two proteins. We use this method to make subunit contact predictions for an additional 36 protein complexes with unknown structures, and present models based on these predictions for the tripartite ATP-independent periplasmic (TRAP) transporter, the tripartite efflux system, the pyruvate formate lyase-activating enzyme complex, and the methionine ABC transporter.DOI: http://dx.doi.org/10.7554/eLife.02030.001.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Do the amino acid sequence identities of residues that make contact across protein interfaces covary during evolution? If so, such covariance could be used to predict contacts across interfaces and assemble models of biological complexes. We find that residue pairs identified using a pseudo-likelihood-based method to covary across protein-protein interfaces in the 50S ribosomal unit and 28 additional bacterial protein complexes with known structure are almost always in contact in the complex, provided that the number of aligned sequences is greater than the average length of the two proteins. We use this method to make subunit contact predictions for an additional 36 protein complexes with unknown structures, and present models based on these predictions for the tripartite ATP-independent periplasmic (TRAP) transporter, the tripartite efflux system, the pyruvate formate lyase-activating enzyme complex, and the methionine ABC transporter.DOI: http://dx.doi.org/10.7554/eLife.02030.001. |
Rajagopalan, Sridharan; Wang, Chu; Yu, Kai; Kuzin, Alexandre P; Richter, Florian; Lew, Scott; Miklos, Aleksandr E; Matthews, Megan L; Seetharaman, Jayaraman; Su, Min; Hunt, John F; Cravatt, Benjamin F; Baker, David Design of activated serine-containing catalytic triads with atomic-level accuracy Journal Article Nature chemical biology, 10 , pp. 386-391, 2014, ISSN: 1552-4469. @article{528, title = {Design of activated serine-containing catalytic triads with atomic-level accuracy}, author = { Sridharan Rajagopalan and Chu Wang and Kai Yu and Alexandre P Kuzin and Florian Richter and Scott Lew and Aleksandr E Miklos and Megan L Matthews and Jayaraman Seetharaman and Min Su and John F Hunt and Benjamin F Cravatt and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Rajagopalan_nchembio_2014A.pdf}, doi = {10.1038/nchembio.1498}, issn = {1552-4469}, year = {2014}, date = {2014-04-01}, journal = {Nature chemical biology}, volume = {10}, pages = {386-391}, abstract = {A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents.}, keywords = {}, pubstate = {published}, tppubtype = {article} } A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents. |
Baker, David Protein folding, structure prediction and design. Journal Article Biochemical Society transactions, 42 , pp. 225-9, 2014, ISSN: 1470-8752. @article{529, title = {Protein folding, structure prediction and design.}, author = { David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Baker_BiochemSocTrans_2014.pdf}, doi = {10.1042/BST20130055}, issn = {1470-8752}, year = {2014}, date = {2014-04-01}, journal = {Biochemical Society transactions}, volume = {42}, pages = {225-9}, abstract = {I describe how experimental studies of protein folding have led to advances in protein structure prediction and protein design. I describe the finding that protein sequences are not optimized for rapid folding, the contact order-protein folding rate correlation, the incorporation of experimental insights into protein folding into the Rosetta protein structure production methodology and the use of this methodology to determine structures from sparse experimental data. I then describe the inverse problem (protein design) and give an overview of recent work on designing proteins with new structures and functions. I also describe the contributions of the general public to these efforts through the Rosetta@home distributed computing project and the FoldIt interactive protein folding and design game.}, keywords = {}, pubstate = {published}, tppubtype = {article} } I describe how experimental studies of protein folding have led to advances in protein structure prediction and protein design. I describe the finding that protein sequences are not optimized for rapid folding, the contact order-protein folding rate correlation, the incorporation of experimental insights into protein folding into the Rosetta protein structure production methodology and the use of this methodology to determine structures from sparse experimental data. I then describe the inverse problem (protein design) and give an overview of recent work on designing proteins with new structures and functions. I also describe the contributions of the general public to these efforts through the Rosetta@home distributed computing project and the FoldIt interactive protein folding and design game. |