Publications
Preprints available on bioRxiv.
Yakov Kipnis Brian D. Weitzner, A. Gerard Daniel
A computational method for design of connected catalytic networks in proteins Journal Article
In: Protein Science, 2019.
@article{Weitzner2019,
title = {A computational method for design of connected catalytic networks in proteins},
author = {Brian D. Weitzner, Yakov Kipnis, A. Gerard Daniel, Donald Hilvert, David Baker},
url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/pro.3757
https://www.bakerlab.org/wp-content/uploads/2020/02/Weitzner_et_al-2019-Protein_Science-1.pdf},
doi = {DOI10.1002/pro .3757},
year = {2019},
date = {2019-10-23},
journal = {Protein Science},
abstract = {Computational design of new active sites has generally proceeded by geometrically defining interactions between the reaction transition state(s) and surrounding side-chain functional groups which maximize transition-state stabilization, and then searching for sites in protein scaffolds where the specified side-chain–transition-state interactions can be realized. A limitation of this approach is that the interactions between the side chains themselves are not constrained. An extensive connected hydrogen bond network involving the catalytic residues was observed in a designed retroaldolase following directed evolution. Such connected networks could increase catalytic activity by preorganizing active site residues in catalytically competent orientations, and enabling concerted interactions between side chains during catalysis, for example proton shuffling. We developed a method for designing active sites in which the catalytic side chains, in addition to making interactions with the transition state, are also involved in extensive hydrogen bond networks. Because of the added constraint of hydrogen-bond connectivity between the catalytic side chains, to find solutions, a wider range of interactions between these side chains and the transition state must be considered. Our new method starts from a ChemDraw-like 2D representation of the transition state with hydrogen-bond donors, acceptors, and covalent interaction sites indicated, and all placements of side-chain functional groups that make the indicated interactions with the transition state, and are fully connected in a single hydrogen-bond network are systematically enumerated. The RosettaMatch method can then be used to identify realizations of these fully-connected active sites in protein scaffolds. The method generates many fully-connected active site solutions for a set of model reactions that are promising starting points for the design of fully-preorganized enzyme catalysts.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Romero Romero, Maria Luisa; Yang, Fan; Lin, Yu-Ru; Toth-Petroczy, Agnes; Berezovsky, Igor N.; Goncearenco, Alexander; Yang, Wen; Wellner, Alon; Kumar-Deshmukh, Fanindra; Sharon, Michal; Baker, David; Varani, Gabriele; Tawfik, Dan S.
Simple yet functional phosphate-loop proteins Journal Article
In: PNAS, vol. 115, no. 51, pp. E11943–E11950, 2018, ISSN: 0027-8424.
@article{Romero2018,
title = {Simple yet functional phosphate-loop proteins},
author = {Romero Romero, Maria Luisa and Yang, Fan and Lin, Yu-Ru and Toth-Petroczy, Agnes and Berezovsky, Igor N. and Goncearenco, Alexander and Yang, Wen and Wellner, Alon and Kumar-Deshmukh, Fanindra and Sharon, Michal and Baker, David and Varani, Gabriele and Tawfik, Dan S.},
url = {https://www.bakerlab.org/wp-content/uploads/2019/02/Romero2018.pdfhttps://www.pnas.org/content/115/51/E11943
},
doi = {10.1073/pnas.1812400115},
issn = {0027-8424},
year = {2018},
date = {2018-11-18},
journal = {PNAS},
volume = {115},
number = {51},
pages = {E11943--E11950},
abstract = {The complexity of modern proteins makes the understanding of how proteins evolved from simple beginnings a daunting challenge. The Walker-A motif is a phosphate-binding loop (P-loop) found in possibly the most ancient and abundant protein class, so-called P-loop NTPases. By combining phylogenetic analysis and computational protein design, we have generated simple proteins, of only 55 residues, that contain the P-loop and thereby confer binding of a range of phosphate-containing ligands{textemdash}and even more avidly, RNA and single-strand DNA. Our results show that biochemical function can be implemented in small and simple proteins; they intriguingly suggest that the P-loop emerged as a polynucleotide binder and catalysis of phosphoryl transfer evolved later upon acquisition of higher sequence and structural complexity.Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein{textquoteright}s scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a β-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed β-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop{textquoteright}s key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple β-α repeat proteins, primarily as a polynucleotide binding motif.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Yue-Ting K. Lau,; Vladimir Baytshtok,; Tessa A. Howard,; Brooke M. Fiala,; JayLee M. Johnson,; Lauren P. Carter,; David Baker,; Christopher D. Lima,; Bahl, Christopher D.
Discovery and engineering of enhanced SUMO protease enzymes Journal Article
In: The Journal of Biological Chemistry, vol. 293, pp. 13224-13233, 2018.
@article{Lau2018,
title = {Discovery and engineering of enhanced SUMO protease enzymes},
author = {Yue-Ting K. Lau, and Vladimir Baytshtok, and Tessa A. Howard, and Brooke M. Fiala, and JayLee M. Johnson, and Lauren P. Carter, and David Baker, and Christopher D. Lima, and Christopher D. Bahl},
url = {http://www.jbc.org/content/293/34/13224.short
https://www.bakerlab.org/wp-content/uploads/2019/02/Lau2018.pdf},
doi = {10.1074/jbc.RA118.004146},
year = {2018},
date = {2018-07-05},
journal = {The Journal of Biological Chemistry},
volume = {293},
pages = {13224-13233},
abstract = {Small ubiquitin-like modifier (SUMO) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a SUMO-specific protease is then used to remove SUMO from these proteins. Although this protease is highly specific, its limited solubility and stability hamper its utility as an in vitro reagent. Here, we report improved SUMO protease enzymes obtained via two approaches. First, we developed a computational method and used it to re-engineer WT Ulp1 from Saccharomyces cerevisiae to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus Chaetomium thermophilum, as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in Escherichia coli, we found that these re-engineered enzymes can be more thermostable and up to 12 times more soluble, all while retaining WT-or-better levels of SUMO protease activity. The computational method we developed to design solubility-enhancing substitutions is based on the RosettaScripts application for the macromolecular modeling suite Rosetta, and it is broadly applicable for the improvement of solution properties of other proteins. Moreover, we determined the X-ray crystal structure of a SUMO protease from C. thermophilum to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the S. cerevisiae SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from C. thermophilum, we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Lu, Peilong; Min, Duyoung; DiMaio, Frank; Wei, Kathy Y.; Vahey, Michael D.; Boyken, Scott E.; Chen, Zibo; Fallas, Jorge A.; Ueda, George; Sheffler, William; Mulligan, Vikram Khipple; Xu, Wenqing; Bowie, James U.; Baker, David
Accurate computational design of multipass transmembrane proteins Journal Article
In: Science, vol. 359, no. 6379, pp. 1042–1046, 2018, ISSN: 0036-8075.
@article{Lu1042,
title = {Accurate computational design of multipass transmembrane proteins},
author = {Lu, Peilong and Min, Duyoung and DiMaio, Frank and Wei, Kathy Y. and Vahey, Michael D. and Boyken, Scott E. and Chen, Zibo and Fallas, Jorge A. and Ueda, George and Sheffler, William and Mulligan, Vikram Khipple and Xu, Wenqing and Bowie, James U. and Baker, David},
url = {http://science.sciencemag.org/content/359/6379/1042
https://www.bakerlab.org/wp-content/uploads/2018/03/Lu_Science_2018.pdf},
doi = {10.1126/science.aaq1739},
issn = {0036-8075},
year = {2018},
date = {2018-03-02},
journal = {Science},
volume = {359},
number = {6379},
pages = {1042--1046},
abstract = {In recent years, soluble protein design has achieved successes such as artificial enzymes and large protein cages. Membrane proteins present a considerable design challenge, but here too there have been advances, including the design of a zinc-transporting tetramer. Lu et al. report the design of stable transmembrane monomers, homodimers, trimers, and tetramers with up to eight membrane-spanning regions in an oligomer. The designed proteins adopted the target oligomerization state and localized to the predicted cellular membranes, and crystal structures of the designed dimer and tetramer reflected the design models.Science, this issue p. 1042The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer{textemdash}a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices{textemdash}are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Marcos, Enrique*; Basanta, Benjamin*; Chidyausiku, Tamuka M.; Tang, Yuefeng; Oberdorfer, Gustav; Liu, Gaohua; Swapna, G. V. T.; Guan, Rongjin; Silva, Daniel-Adriano; Dou, Jiayi; Pereira, Jose Henrique; Xiao, Rong; Sankaran, Banumathi; Zwart, Peter H.; Montelione, Gaetano T.; Baker, David
Principles for designing proteins with cavities formed by curved β sheets Journal Article
In: Science, vol. 355, no. 6321, pp. 201–206, 2017, ISSN: 0036-8075.
@article{Marcos2017,
title = {Principles for designing proteins with cavities formed by curved β sheets},
author = {Marcos, Enrique* and Basanta, Benjamin* and Chidyausiku, Tamuka M. and Tang, Yuefeng and Oberdorfer, Gustav and Liu, Gaohua and Swapna, G. V. T. and Guan, Rongjin and Silva, Daniel-Adriano and Dou, Jiayi and Pereira, Jose Henrique and Xiao, Rong and Sankaran, Banumathi and Zwart, Peter H. and Montelione, Gaetano T. and Baker, David},
url = {https://www.bakerlab.org/wp-content/uploads/2017/01/Marcos_Science_2017.pdf
http://science.sciencemag.org/content/355/6321/201},
doi = {10.1126/science.aah7389},
issn = {0036-8075},
year = {2017},
date = {2017-01-01},
journal = {Science},
volume = {355},
number = {6321},
pages = {201--206},
publisher = {American Association for the Advancement of Science},
abstract = {In de novo protein design, creating custom-tailored binding sites is a particular challenge because these sites often involve nonideal backbone structures. For example, curved b sheets are a common ligand binding motif. Marcos et al. investigated the principles that drive β-sheet curvature by studying the geometry of β sheets in natural proteins and folding simulations. In a step toward custom design of enzyme catalysts, they used these principles to control β-sheet geometry and design proteins with differently shaped cavities.Science, this issue p. 201Active sites and ligand-binding cavities in native proteins are often formed by curved β sheets, and the ability to control β-sheet curvature would allow design of binding proteins with cavities customized to specific ligands. Toward this end, we investigated the mechanisms controlling β-sheet curvature by studying the geometry of β sheets in naturally occurring protein structures and folding simulations. The principles emerging from this analysis were used to design, de novo, a series of proteins with curved β sheets topped with α helices. Nuclear magnetic resonance and crystal structures of the designs closely match the computational models, showing that β-sheet curvature can be controlled with atomic-level accuracy. Our approach enables the design of proteins with cavities and provides a route to custom design ligand-binding and catalytic sites.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Bale, Jacob B.; Gonen, Shane; Liu, Yuxi; Sheffler, William; Ellis, Daniel; Thomas, Chantz; Cascio, Duilio; Yeates, Todd O.; Gonen, Tamir; King, Neil P.; Baker, David
Accurate design of megadalton-scale two-component icosahedral protein complexes Journal Article
In: Science, vol. 353, no. 6297, pp. 389-394, 2016.
@article{Bale2016,
title = {Accurate design of megadalton-scale two-component icosahedral protein complexes},
author = {Jacob B. Bale and Shane Gonen and Yuxi Liu and William Sheffler and Daniel Ellis and Chantz Thomas and Duilio Cascio and Todd O. Yeates and Tamir Gonen and Neil P. King and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/07/Bale_Science_2016.pdf},
doi = {10.1126/science.aaf8818},
year = {2016},
date = {2016-07-22},
journal = {Science},
volume = {353},
number = {6297},
pages = {389-394},
abstract = {Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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
In: Nature, vol. 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}
}
Goldsmith, M; Eckstein, S; Ashani, Y; Greisen, P Jr; 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
In: 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}
}
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
In: Nature Chemical Biology, vol. 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}
}
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
In: 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}
}
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
In: 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}
}
Vittal, Vinayak; Shi, Lei; Wenzel, Dawn M; Scaglione, K Matthew; 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
In: Nature Chemical Biology, vol. 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}
}
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
In: Proceedings of the National Academy of Sciences of the United States of America, vol. 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}
}
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
In: Journal of the American Chemical Society, vol. 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}
}
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
In: Proceedings of the National Academy of Sciences of the United States of America, vol. 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}
}
Ovchinnikov, Sergey; Kamisetty, Hetunandan; Baker, David
Robust and accurate prediction of residue-residue interactions across protein interfaces using evolutionary information. Journal Article
In: eLife, vol. 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}
}
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
In: Nature chemical biology, vol. 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}
}
Wang, Yupeng; Khan, Iram F; Boissel, Sandrine; Jarjour, Jordan; Pangallo, Joseph; Thyme, Summer; Baker, David; Scharenberg, Andrew M; Rawlings, David J
Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus. Journal Article
In: Nucleic acids research, 2014, ISSN: 1362-4962.
@article{527,
title = {Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus.},
author = { Yupeng Wang and Iram F Khan and Sandrine Boissel and Jordan Jarjour and Joseph Pangallo and Summer Thyme and David Baker and Andrew M Scharenberg and David J Rawlings},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Boissel_NucleicAcids_2014.pdf},
issn = {1362-4962},
year = {2014},
date = {2014-03-01},
journal = {Nucleic acids research},
abstract = {LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20-22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)-a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Liu, Yu; Tan, Yun Lei; Zhang, Xin; Bhabha, Gira; Ekiert, Damian C; Genereux, Joseph C; Cho, Younhee; Kipnis, Yakov; Bjelic, Sinisa; Baker, David; Kelly, Jeffery W
Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts. Journal Article
In: Proceedings of the National Academy of Sciences of the United States of America, 2014, ISSN: 1091-6490.
@article{526,
title = {Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts.},
author = { Yu Liu and Yun Lei Tan and Xin Zhang and Gira Bhabha and Damian C Ekiert and Joseph C Genereux and Younhee Cho and Yakov Kipnis and Sinisa Bjelic and David Baker and Jeffery W Kelly},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Liu_PNAS_2014.pdf},
issn = {1091-6490},
year = {2014},
date = {2014-03-01},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
abstract = {Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a proteintextquoterights folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular proteintextquoterights functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Wijma, Hein J; Floor, Robert J; Jekel, Peter A; Baker, David; Marrink, Siewert J; Janssen, Dick B
Computationally designed libraries for rapid enzyme stabilization. Journal Article
In: Protein engineering, design & selection : PEDS, vol. 27, pp. 49-58, 2014, ISSN: 1741-0134.
@article{520,
title = {Computationally designed libraries for rapid enzyme stabilization.},
author = { Hein J Wijma and Robert J Floor and Peter A Jekel and David Baker and Siewert J Marrink and Dick B Janssen},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Wijma_PEDS_2014.pdf},
doi = {10.1093/protein/gzt061},
issn = {1741-0134},
year = {2014},
date = {2014-02-01},
journal = {Protein engineering, design & selection : PEDS},
volume = {27},
pages = {49-58},
abstract = {The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10-12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85textdegreeC, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2024
FROM THE LAB
Sorry, no publications matched your criteria.
COLLABORATOR LED
Sorry, no publications matched your criteria.
2023
FROM THE LAB
Sorry, no publications matched your criteria.
COLLABORATOR LED
Sorry, no publications matched your criteria.
2022
FROM THE LAB
Sorry, no publications matched your criteria.
COLLABORATOR LED
Sorry, no publications matched your criteria.
2021
FROM THE LAB
Sorry, no publications matched your criteria.
COLLABORATOR LED
Sorry, no publications matched your criteria.
2020
FROM THE LAB
Sorry, no publications matched your criteria.
COLLABORATOR LED
Sorry, no publications matched your criteria.
2019
FROM THE LAB
Sorry, no publications matched your criteria.
COLLABORATOR LED
Sorry, no publications matched your criteria.
2018
FROM THE LAB
Sorry, no publications matched your criteria.
COLLABORATOR LED
Sorry, no publications matched your criteria.
2017-1988
ALL PAPERS
Sorry, no publications matched your criteria.