@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@article{510,

title = {Amyloid β peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aβ-mediated toxicity in vitro.},

author = { Tyler D Shropshire and Jack Reifert and Sridharan Rajagopalan and David Baker and Stuart C Feinstein and Patrick S Daugherty},

doi = {10.1515/hsz-2013-0230},

issn = {1437-4315},

year  = {2014},

date = {2014-01-01},

journal = {Biological chemistry},

volume = {395},

pages = {109-18},

abstract = {Abstract The gradual accumulation and assembly of β-amyloid (Aβ) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aβ is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aβ-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)textdownarrow(Y/F)textdownarrow(S/A/G/T) or RXΦ(Y/F)textdownarrow(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aβ (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aβ42 in vitro, thereby inhibiting Aβ fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aβ oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aβ42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aβ-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aβ-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{525,

title = {Redesigning the Specificity of Protein-DNA Interactions with Rosetta.},

author = { Summer Thyme and David Baker},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Thyme_2014.pdf},

doi = {10.1007/978-1-62703-968-0_17},

issn = {1940-6029},

year  = {2014},

date = {2014-01-01},

journal = {Methods in molecular biology (Clifton, N.J.)},

volume = {1123},

pages = {265-82},

abstract = {Building protein tools that can selectively bind or cleave specific DNA sequences requires efficient technologies for modifying protein-DNA interactions. Computational design is one method for accomplishing this goal. In this chapter, we present the current state of protein-DNA interface design with the Rosetta macromolecular modeling program. The LAGLIDADG endonuclease family of DNA-cleaving enzymes, under study as potential gene therapy reagents, has been the main testing ground for these in silico protocols. At this time, the computational methods are most useful for designing endonuclease variants that can accommodate small numbers of target site substitutions. Attempts to engineer for more extensive interface changes will likely benefit from an approach that uses the computational design results in conjunction with a high-throughput directed evolution or screening procedure. The family of enzymes presents an engineering challenge because their interfaces are highly integrated and there is significant coordination between the binding and catalysis events. Future developments in the computational algorithms depend on experimental feedback to improve understanding and modeling of these complex enzymatic features. This chapter presents both the basic method of design that has been successfully used to modulate specificity and more advanced procedures that incorporate DNA flexibility and other properties that are likely necessary for reliable modeling of more extensive target site changes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{497,

title = {Engineering V-type nerve agents detoxifying enzymes using computationally focused libraries},

author = { Izhack Cherny and Per Greisen and Yacov Ashani and Sagar D Khare and Gustav Oberdorfer and Haim Leader and David Baker and Dan S Tawfik},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Cherny_cb4004892_13W.pdf},

doi = {10.1021/cb4004892},

issn = {1554-8937},

year  = {2013},

date = {2013-11-01},

journal = {ACS chemical biology},

volume = {8},

pages = {2394-403},

abstract = {VX and its Russian (RVX) and Chinese (CVX) analogues rapidly inactivate acetylcholinesterase and are the most toxic stockpile nerve agents. These organophosphates have a thiol leaving group with a choline-like moiety and are hydrolyzed very slowly by natural enzymes. We used an integrated computational and experimental approach to increase Brevundimonas diminuta phosphotriesterasetextquoterights (PTE) detoxification rate of V-agents by 5000-fold. Computational models were built of the complex between PTE and V-agents. On the basis of these models, the active site was redesigned to be complementary in shape to VX and RVX and to include favorable electrostatic interactions with their choline-like leaving group. Small libraries based on designed sequences were constructed. The libraries were screened by a direct assay for V-agent detoxification, as our initial studies showed that colorimetric surrogates fail to report the detoxification rates of the actual agents. The experimental results were fed back to improve the computational models. Overall, five rounds of iterating between experiment and model refinement led to variants that hydrolyze the toxic SP isomers of all three V-agents with kcat/KM values of up to 5 texttimes 10(6) M(-1) min(-1) and also efficiently detoxify G-agents. These new catalysts provide the basis for broad spectrum nerve agent detoxification.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{495,

title = {Automating human intuition for protein design},

author = { Lucas G Niv'on and Sinisa Bjelic and Chris King and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Nivón_prot24463_13M.pdf},

doi = {10.1002/prot.24463},

issn = {1097-0134},

year  = {2013},

date = {2013-10-01},

journal = {Proteins},

abstract = {In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer-generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one-position-at-a-time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins. Proteins 2013. textcopyright 2013 Wiley Periodicals, Inc.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{511,

title = {Computational design of a protein-based enzyme inhibitor.},

author = { Erik Procko and Rickard Hedman and Keith Hamilton and Jayaraman Seetharaman and Sarel J Fleishman and Min Su and James Aramini and Gregory Kornhaber and John F Hunt and Liang Tong and Gaetano T Montelione and David Baker},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Procko13.pdf},

doi = {10.1016/j.jmb.2013.06.035},

issn = {1089-8638},

year  = {2013},

date = {2013-09-01},

journal = {Journal of molecular biology},

volume = {425},

pages = {3563-75},

abstract = {While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{509,

title = {Computational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy.},

author = { Jeremy H Mills and Sagar D Khare and Jill M Bolduc and Farhad Forouhar and Vikram Khipple Mulligan and Scott Lew and Jayaraman Seetharaman and Liang Tong and Barry L Stoddard and David Baker},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Mills13.pdf},

doi = {10.1021/ja403503m},

issn = {1520-5126},

year  = {2013},

date = {2013-09-01},

journal = {Journal of the American Chemical Society},

volume = {135},

pages = {13393-9},

abstract = {Genetically encoded unnatural amino acids could facilitate the design of proteins and enzymes of novel function, but correctly specifying sites of incorporation and the identities and orientations of surrounding residues represents a formidable challenge. Computational design methods have been used to identify optimal locations for functional sites in proteins and design the surrounding residues but have not incorporated unnatural amino acids in this process. We extended the Rosetta design methodology to design metalloproteins in which the amino acid (2,2textquoteright-bipyridin-5yl)alanine (Bpy-Ala) is a primary ligand of a bound metal ion. Following initial results that indicated the importance of buttressing the Bpy-Ala amino acid, we designed a buried metal binding site with octahedral coordination geometry consisting of Bpy-Ala, two protein-based metal ligands, and two metal-bound water molecules. Experimental characterization revealed a Bpy-Ala-mediated metalloprotein with the ability to bind divalent cations including Co(2+), Zn(2+), Fe(2+), and Ni(2+), with a Kd for Zn(2+) of ~40 pM. X-ray crystal structures of the designed protein bound to Co(2+) and Ni(2+) have RMSDs to the design model of 0.9 and 1.0 r A respectively over all atoms in the binding site.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{504,

title = {Evolution of a designed retro-aldolase leads to complete active site remodeling.},

author = { Lars Giger and Sami Caner and Richard Obexer and Peter Kast and David Baker and Nenad Ban and Donald Hilvert},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Giger_nchembio_2013.pdf},

doi = {10.1038/nchembio.1276},

issn = {1552-4469},

year  = {2013},

date = {2013-08-01},

journal = {Nature chemical biology},

volume = {9},

pages = {494-8},

abstract = {Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that pronounced molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400-fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate-binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{472,

title = {Computational enzyme design},

author = { Gert Kiss and Nihan Celebi-"Olc c"um and Rocco Moretti and David Baker and K N Houk},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Kiss_AngewChemIntEd_2013.pdf},

doi = {10.1002/anie.201204077},

issn = {1521-3773},

year  = {2013},

date = {2013-05-01},

journal = {Angewandte Chemie (International ed. in English)},

volume = {52},

pages = {5700-25},

abstract = {Recent developments in computational chemistry and biology have come together in the "inside-out" approach to enzyme engineering. Proteins have been designed to catalyze reactions not previously accelerated in nature. Some of these proteins fold and act as catalysts, but the success rate is still low. The achievements and limitations of the current technology are highlighted and contrasted to other protein engineering techniques. On its own, computational "inside-out" design can lead to the production of catalytically active and selective proteins, but their kinetic performances fall short of natural enzymes. When combined with directed evolution, molecular dynamics simulations, and crowd-sourced structure-prediction approaches, however, computational designs can be significantly improved in terms of binding, turnover, and thermal stability.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{503,

title = {Expanding the product profile of a microbial alkane biosynthetic pathway.},

author = { Matthew Harger and Lei Zheng and Austin Moon and Casey Ager and Ju Hye An and Chris Choe and Yi-Ling Lai and Benjamin Mo and David Zong and Matthew D Smith and Robert G Egbert and Jeremy H Mills and David Baker and Ingrid Swanson Pultz and Justin B Siegel},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Harger_ACSSynthBiol_2013.pdf},

doi = {10.1021/sb300061x},

issn = {2161-5063},

year  = {2013},

date = {2013-01-01},

journal = {ACS synthetic biology},

volume = {2},

pages = {59-62},

abstract = {Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis , an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli . When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{470,

title = {A Pareto-optimal refinement method for protein design scaffolds},

author = { Lucas Gregorio Niv'on and Rocco Moretti and David Baker},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Nivon_pone0059004_13U.pdf},

doi = {10.1371/journal.pone.0059004},

issn = {1932-6203},

year  = {2013},

date = {2013-00-01},

journal = {PloS one},

volume = {8},

pages = {e59004},

abstract = {Computational design of protein function involves a search for amino acids with the lowest energy subject to a set of constraints specifying function. In many cases a set of natural protein backbone structures, or "scaffolds", are searched to find regions where functional sites (an enzyme active site, ligand binding pocket, protein-protein interaction region, etc.) can be placed, and the identities of the surrounding amino acids are optimized to satisfy functional constraints. Input native protein structures almost invariably have regions that score very poorly with the design force field, and any design based on these unmodified structures may result in mutations away from the native sequence solely as a result of the energetic strain. Because the input structure is already a stable protein, it is desirable to keep the total number of mutations to a minimum and to avoid mutations resulting from poorly-scoring input structures. Here we describe a protocol using cycles of minimization with combined backbone/sidechain restraints that is Pareto-optimal with respect to RMSD to the native structure and energetic strain reduction. The protocol should be broadly useful in the preparation of scaffold libraries for functional site design.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{479,

title = {Computational Design of an α-gliadin Peptidase},

author = { Sydney R Gordon and Elizabeth J Stanley and Sarah Wolf and Angus Toland and Sean J Wu and Daniel Hadidi and Jeremy H Mills and David Baker and Ingrid Swanson Pultz and Justin B Siegel},

url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Gordon12E.pdf

http://www.ncbi.nlm.nih.gov/pubmed/23153249},

doi = {10.1021/ja3094795},

issn = {1520-5126},

year  = {2012},

date = {2012-12-01},

journal = {Journal of the American Chemical Society},

volume = {134},

pages = {20513-20},

abstract = {The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{452,

title = {Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.},

author = { Florian Richter and Rebecca Blomberg and Sagar D Khare and Gert Kiss and Alexandre P Kuzin and Adam J T Smith and Jasmine Gallaher and Zbigniew Pianowski and Roger C Helgeson and Alexej Grjasnow and Rong Xiao and Jayaraman Seetharaman and Min Su and Sergey Vorobiev and Scott Lew and Farhad Forouhar and Gregory J Kornhaber and John F Hunt and Gaetano T Montelione and Liang Tong and K N Houk and Donald Hilvert and David Baker},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Richter_JACS_2012.pdf},

doi = {10.1021/ja3037367},

issn = {1520-5126},

year  = {2012},

date = {2012-10-01},

journal = {Journal of the American Chemical Society},

volume = {134},

pages = {16197-206},

abstract = {Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{455,

title = {Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases.},

author = { Sarah Baxter and Abigail R Lambert and Ryan Kuhar and Jordan Jarjour and Nadia Kulshina and Fabio Parmeggiani and Patrick Danaher and Jacob Gano and David Baker and Barry L Stoddard and Andrew M Scharenberg},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Baxter_NuclAcRes_2012.pdf},

issn = {1362-4962},

year  = {2012},

date = {2012-09-01},

journal = {Nucleic acids research},

volume = {40},

pages = {7985-8000},

abstract = {Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ~70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{603,

title = {Comparison of designed and randomly generated catalysts for simple chemical reactions},

author = { Yakov Kipnis and David Baker},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/comparisonofdesigned_Baker2012.pdf},

doi = {10.1002/pro.2125},

issn = {1469-896X},

year  = {2012},

date = {2012-09-01},

journal = {Protein Science : A Publication of the Protein Society},

volume = {21},

pages = {1388-95},

abstract = {There has been recent success in designing enzymes for simple chemical reactions using a two-step protocol. In the first step, a geometric matching algorithm is used to identify naturally occurring protein scaffolds at which predefined idealized active sites can be realized. In the second step, the residues surrounding the transition state model are optimized to increase transition state binding affinity and to bolster the primary catalytic side chains. To improve the design methodology, we investigated how the set of solutions identified by the design calculations relate to the overall set of solutions for two different chemical reactions. Using a TIM barrel scaffold in which catalytically active Kemp eliminase and retroaldolase designs were obtained previously, we carried out activity screens of random libraries made to be compositionally similar to active designs. A small number of active catalysts were found in screens of 10textthreesuperior variants for each of the two reactions, which differ from the computational designs in that they reuse charged residues already present in the native scaffold. The results suggest that computational design considerably increases the frequency of catalyst generation for active sites involving newly introduced catalytic residues, highlighting the importance of interaction cooperativity in enzyme active sites.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{600,

title = {The dynamic disulphide relay of quiescin sulphydryl oxidase},

author = { Assaf Alon and Iris Grossman and Yair Gat and Vamsi K Kodali and Frank DiMaio and Tevie Mehlman and Gilad Haran and David Baker and Colin Thorpe and Deborah Fass},

url = {https://www.bakerlab.org/wp-content/uploads/2016/01/thedynamicdisulphide_Baker2012.pdf},

doi = {10.1038/nature11267},

issn = {1476-4687},

year  = {2012},

date = {2012-08-01},

journal = {Nature},

volume = {488},

pages = {414-8},

abstract = {Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 r A apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165textdegree domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{456,

title = {Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59.},

author = { Olga Khersonsky and Gert Kiss and Daniela R"othlisberger and Orly Dym and Shira Albeck and Kendall N Houk and David Baker and Dan S Tawfik},

url = {https://www.bakerlab.org/wp-content/uploads/2018/06/10358.full_.pdf

www.pnas.org/content/109/26/10358},

issn = {1091-6490},

year  = {2012},

date = {2012-06-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {109},

pages = {10358-63},

abstract = {Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k(cat)/K(m) values of <= 5 10(4) M(-1)s(-1)). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k(cat)/K(m), and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59textquoterights evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to > 2,000-fold increase in catalytic efficiency, mainly via higher k(cat) values. The best KE59 variants exhibited k(cat)/K(m) values up to 0.6 10(6) M(-1)s(-1), and k(cat)/k(uncat) values of <= 10(7) almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{461,

title = {Robust design and optimization of retroaldol enzymes.},

author = { Eric A Althoff and Ling Wang and Lin Jiang and Lars Giger and Jonathan K Lassila and Zhizhi Wang and Matthew Smith and Sanjay Hari and Peter Kast and Daniel Herschlag and Donald Hilvert and David Baker},

url = {http://beta.baker/wp-content/uploads/2015/12/Althoff_ProteinScience_2012.pdf},

doi = {10.1002/pro.2059},

issn = {1469-896X},

year  = {2012},

date = {2012-05-01},

journal = {Protein science : a publication of the Protein Society},

volume = {21},

pages = {717-26},

abstract = {Enzyme catalysts of a retroaldol reaction have been generated by computational design using a motif that combines a lysine in a nonpolar environment with water-mediated stabilization of the carbinolamine hydroxyl and β-hydroxyl groups. Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{427,

title = {Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis},

author = { Sagar D Khare and Yakov Kipnis and Per Jr Greisen and Ryo Takeuchi and Yacov Ashani and Moshe Goldsmith and Yifan Song and Jasmine L Gallaher and Israel Silman and Haim Leader and Joel L Sussman and Barry L Stoddard and Dan S Tawfik and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2018/06/nchembio.777.pdf

https://www.nature.com/articles/nchembio.777},

doi = {10.1038/nchembio.777},

issn = {1552-4469},

year  = {2012},

date = {2012-02-01},

journal = {Nature chemical biology},

abstract = {The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{431,

title = {An engineered microbial platform for direct biofuel production from brown macroalgae},

author = { Adam J Wargacki and Effendi Leonard and Maung Nyan Win and Drew D Regitsky and Christine Nicole S Santos and Peter B Kim and Susan R Cooper and Ryan M Raisner and Asael Herman and Alicia B Sivitz and Arun Lakshmanaswamy and Yuki Kashiyama and David Baker and Yasuo Yoshikuni},

url = {https://www.bakerlab.org/wp-content/uploads/2018/06/308.full_.pdf

http://science.sciencemag.org/content/335/6066/308},

doi = {10.1126/science.1214547 },

issn = {1095-9203},

year  = {2012},

date = {2012-01-01},

journal = {Science},

volume = {335},

pages = {308-13},

abstract = {Prospecting macroalgae (seaweeds) as feedstocks for bioconversion into biofuels and commodity chemical compounds is limited primarily by the availability of tractable microorganisms that can metabolize alginate polysaccharides. Here, we present the discovery of a 36-kilo-base pair DNA fragment from Vibrio splendidus encoding enzymes for alginate transport and metabolism. The genomic integration of this ensemble, together with an engineered system for extracellular alginate depolymerization, generated a microbial platform that can simultaneously degrade, uptake, and metabolize alginate. When further engineered for ethanol synthesis, this platform enables bioethanol production directly from macroalgae via a consolidated process, achieving a titer of 4.7% volume/volume and a yield of 0.281 weight ethanol/weight dry macroalgae (equivalent to ~80% of the maximum theoretical yield from the sugar composition in macroalgae).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{430,

title = {Structural analyses of covalent enzyme-substrate analog complexes reveal strengths and limitations of de novo enzyme design},

author = { Ling Wang and Eric A Althoff and Jill Bolduc and Lin Jiang and James Moody and Jonathan K Lassila and Lars Giger and Donald Hilvert and Barry Stoddard and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2018/06/1-s2.0-S0022283611011910-main.pdf

https://www.sciencedirect.com/science/article/pii/S0022283611011910?via%3Dihub},

doi = {10.1016/j.jmb.2011.10.043},

issn = {1089-8638},

year  = {2012},

date = {2012-01-01},

journal = {Journal of molecular biology},

volume = {415},

pages = {615-25},

abstract = {We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with a covalently bound mechanism-based inhibitor is similar to, but not identical with, the design model, with an RMSD of 1.4~r A over active-site residues and equivalent substrate atoms. As in the design model, the binding pocket orients the substrate through hydrophobic interactions with the naphthyl moiety such that the oxygen atoms analogous to the carbinolamine and β-hydroxyl oxygens are positioned near a network of bound waters. However, there are differences between the design model and the structure: the orientation of the naphthyl group and the conformation of the catalytic lysine are slightly different; the bound water network appears to be more extensive; and the bound substrate analog exhibits more conformational heterogeneity than typical native enzyme-inhibitor complexes. Alanine scanning of the active-site residues shows that both the catalytic lysine and the residues around the binding pocket for the substrate naphthyl group make critical contributions to catalysis. Mutating the set of water-coordinating residues also significantly reduces catalytic activity. The crystal structure of the enzyme with a smaller substrate analog that lacks naphthyl ring shows the catalytic lysine to be more flexible than in the naphthyl-substrate complex; increased preorganization of the active site would likely improve catalysis. The covalently bound complex structures and mutagenesis data highlight the strengths and weaknesses of the de novo enzyme design strategy.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{434,

title = {Increased Diels-Alderase activity through backbone remodeling guided by Foldit players},

author = { Christopher B Eiben and Justin B Siegel and Jacob B Bale and Seth Cooper and Firas Khatib and Betty W Shen and Foldit Players and Barry L Stoddard and Zoran Popovic and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2018/06/nbt.2109.pdf

https://www.nature.com/articles/nbt.2109},

doi = {10.1038/nbt.2109},

issn = {1546-1696},

year  = {2012},

date = {2012-00-01},

journal = {Nature biotechnology},

volume = {30},

pages = {190-2},

abstract = {Computational enzyme design holds promise for the production of renewable fuels, drugs and chemicals. De novo enzyme design has generated catalysts for several reactions, but with lower catalytic efficiencies than naturally occurring enzymes. Here we report the use of game-driven crowdsourcing to enhance the activity of a computationally designed enzyme through the functional remodeling of its structure. Players of the online game Foldit were challenged to remodel the backbone of a computationally designed bimolecular Diels-Alderase to enable additional interactions with substrates. Several iterations of design and characterization generated a 24-residue helix-turn-helix motif, including a 13-residue insertion, that increased enzyme activity >18-fold. X-ray crystallography showed that the large insertion adopts a helix-turn-helix structure positioned as in the Foldit model. These results demonstrate that human creativity can extend beyond the macroscopic challenges encountered in everyday life to molecular-scale design problems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{436,

title = {Mining endonuclease cleavage determinants in genomic sequence data.},

author = { Mindy D Szeto and Sandrine J S Boissel and David Baker and Summer B Thyme},

url = {https://www.bakerlab.org/wp-content/uploads/2018/06/J.-Biol.-Chem.-2011-Szeto-32617-27.pdf

http://www.jbc.org/content/286/37/32617},

doi = {10.1074/jbc.M111.259572},

issn = {1083-351X},

year  = {2011},

date = {2011-09-01},

journal = {The Journal of biological chemistry},

volume = {286},

pages = {32617-27},

abstract = {Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Richter2011,

title = {De Novo Enzyme Design Using Rosetta3},

author = {Richter, Florian AND Leaver-Fay, Andrew AND Khare, Sagar D. AND Bjelic, Sinisa AND Baker, David

},

url = {https://doi.org/10.1371/journal.pone.0019230},

doi = {10.1371/journal.pone.0019230},

year  = {2011},

date = {2011-05-11},

journal = {PLoS},

abstract = {The Rosetta de novo enzyme design protocol has been used to design enzyme catalysts for a variety of chemical reactions, and in principle can be applied to any arbitrary chemical reaction of interest, The process has four stages: 1) choice of a catalytic mechanism and corresponding minimal model active site, 2) identification of sites in a set of scaffold proteins where this minimal active site can be realized, 3) optimization of the identities of the surrounding residues for stabilizing interactions with the transition state and primary catalytic residues, and 4) evaluation and ranking the resulting designed sequences. Stages two through four of this process can be carried out with the Rosetta package, while stage one needs to be done externally. Here, we demonstrate how to carry out the Rosetta enzyme design protocol from start to end in detail using for illustration the triosephosphate isomerase reaction.



},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{351,

title = {Optimization of the In-Silico-Designed Kemp Eliminase KE70 by Computational Design and Directed Evolution},

author = { Olga Khersonsky and Daniela R"othlisberger and Andrew M Wollacott and Paul Murphy and Orly Dym and Shira Albeck and Gert Kiss and K N Houk and David Baker and Dan S Tawfik},

issn = {1089-8638},

year  = {2011},

date = {2011-04-01},

journal = {Journal of molecular biology},

volume = {407},

pages = {391-412},

abstract = {Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher k(cat) values and lower K(m) values, with the best variants exhibiting k(cat)/K(m) values of >5texttimes10(4)~s(-)(1) M(-1). The optimized KE70 variants were characterized structurally and biochemically, providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active-site cavity to achieve tighter substrate binding; second, the fine-tuning of electrostatics around the catalytic His-Asp dyad; and, third, the stabilization of the active-site dyad in a conformation optimal for catalysis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{355,

title = {Crystal structure of XMRV protease differs from the structures of other retropepsins},

author = { Mi Li and Frank DiMaio and Dongwen Zhou and Alla Gustchina and Jacek Lubkowski and Zbigniew Dauter and David Baker and Alexander Wlodawer},

issn = {1545-9985},

year  = {2011},

date = {2011-02-01},

journal = {Nature structural & molecular biology},

volume = {18},

pages = {227-9},

abstract = {Using energy and density guided Rosetta refinement to improve molecular replacement, we determined the crystal structure of the protease encoded by xenotropic murine leukemia virus-related virus (XMRV). Despite overall similarity of XMRV protease to other retropepsins, the topology of its dimer interface more closely resembles those of the monomeric, pepsin-like enzymes. Thus, XMRV protease may represent a distinct branch of the aspartic protease family.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{268,

title = {Computational design of orthogonal nucleoside kinases},

author = { Lingfeng Liu and Paul Murphy and David Baker and Stefan Lutz},

issn = {1364-548X},

year  = {2010},

date = {2010-12-01},

journal = {Chemical communications},

volume = {46},

pages = {8803-5},

abstract = {We report the computational enzyme design of an orthogonal nucleoside analog kinase for 3textquoteright-deoxythymidine. The best kinase variant shows an 8500-fold change in substrate specificity, resulting from a 4.6-fold gain in catalytic efficiency for the nucleoside analog and a 2000-fold decline for the native substrate thymidine.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{265,

title = {Evaluation and ranking of enzyme designs},

author = { Gert Kiss and Daniela R"othlisberger and David Baker and K N Houk},

doi = {10.1002/pro.462},

issn = {1469-896X},

year  = {2010},

date = {2010-09-01},

journal = {Protein science},

volume = {19},

pages = {1760-73},

abstract = {In 2008, a successful computational design procedure was reported that yielded active enzyme catalysts for the Kemp elimination. Here, we studied these proteins together with a set of previously unpublished inactive designs to determine the sources of activity or lack thereof, and to predict which of the designed structures are most likely to be catalytic. Methods that range from quantum mechanics (QM) on truncated model systems to the treatment of the full protein with ONIOM QM/MM and AMBER molecular dynamics (MD) were explored. The most effective procedure involved molecular dynamics, and a general MD protocol was established. Substantial deviations from the ideal catalytic geometries were observed for a number of designs. Penetration of water into the catalytic site and insufficient residue-packing around the active site are the main factors that can cause enzyme designs to be inactive. Where in the past, computational evaluations of designed enzymes were too time-extensive for practical considerations, it has now become feasible to rank and refine candidates computationally prior to and in conjunction with experimentation, thus markedly increasing the efficiency of the enzyme design process.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{229,

title = {An exciting but challenging road ahead for computational enzyme design},

author = { David Baker},

issn = {1469-896X},

year  = {2010},

date = {2010-08-01},

journal = {Protein science},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{91,

title = {Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction},

author = { Justin B Siegel and Alexandre Zanghellini and Helena M Lovick and Gert Kiss and Abigail R Lambert and Jennifer L St Clair and Jasmine L Gallaher and Donald Hilvert and Michael H Gelb and Barry L Stoddard and Kendall N Houk and Forrest E Michael and David Baker},

issn = {1095-9203},

year  = {2010},

date = {2010-07-01},

journal = {Science},

volume = {329},

pages = {309-13},

abstract = {The Diels-Alder reaction is a cornerstone in organic synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimolecular Diels-Alder reactions. We describe the de novo computational design and experimental characterization of enzymes catalyzing a bimolecular Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallography confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond-forming reactions should be broadly useful in synthetic chemistry.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{583,

title = {Evolutionary optimization of computationally designed enzymes: Kemp eliminases of the KE07 series},

author = { Olga Khersonsky and Daniela R"othlisberger and Orly Dym and Shira Albeck and Colin J Jackson and David Baker and Dan S Tawfik},

doi = {10.1016/j.jmb.2009.12.031},

issn = {1089-8638},

year  = {2010},

date = {2010-03-01},

journal = {Journal of Molecular Biology},

volume = {396},

pages = {1025-42},

abstract = {Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge-their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a >200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07textquoterights activity to give a k(cat)/K(M) value of approximately 2600 s(-1) M(-1) and an approximately 10(6)-fold rate acceleration (k(cat)/k(uncat)) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07textquoterights improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its "quenching" interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pK(a) of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{138,

title = {Exploitation of binding energy for catalysis and design},

author = { Summer B Thyme and Jordan Jarjour and Ryo Takeuchi and James J Havranek and Justin Ashworth and Andrew M Scharenberg and Barry L Stoddard and David Baker},

issn = {1476-4687},

year  = {2009},

date = {2009-10-01},

journal = {Nature},

volume = {461},

pages = {1300-4},

abstract = {Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the centre of a 20-base-pair (bp) DNA target site, with the amino (N)-terminal domain of the enzyme making extensive binding interactions with the left (-) side of the target site and the similarly structured carboxy (C)-terminal domain interacting with the right (+) side. Here we show that, despite the approximate twofold symmetry of the enzyme-DNA complex, there is almost complete segregation of interactions responsible for substrate binding to the (-) side of the interface and interactions responsible for transition-state stabilization to the (+) side. Although single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (-) DNA half-site almost exclusively increase the dissociation constant (K(D)) and the Michaelis constant under single-turnover conditions (K(M)*), and those in the (+) half-site primarily decrease the turnover number (k(cat)*). The reduction of activity produced by mutations on the (-) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the use of enzyme-substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (-) side do so by modulating K(M)*, whereas redesigns with altered specificities on the (+) side modulate k(cat)*. Our results illustrate how classical enzymology and modern protein design can each inform the other.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{133,

title = {Alteration of enzyme specificity by computational loop remodeling and design},

author = { Paul M Murphy and Jill M Bolduc and Jasmine L Gallaher and Barry L Stoddard and David Baker},

issn = {1091-6490},

year  = {2009},

date = {2009-06-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {106},

pages = {9215-20},

abstract = {Altering the specificity of an enzyme requires precise positioning of side-chain functional groups that interact with the modified groups of the new substrate. This requires not only sequence changes that introduce the new functional groups but also sequence changes that remodel the structure of the protein backbone so that the functional groups are properly positioned. We describe a computational design method for introducing specific enzyme-substrate interactions by directed remodeling of loops near the active site. Benchmark tests on 8 native protein-ligand complexes show that the method can recover native loop lengths and, often, native loop conformations. We then use the method to redesign a critical loop in human guanine deaminase such that a key side-chain interaction is made with the substrate ammelide. The redesigned enzyme is 100-fold more active on ammelide and 2.5e4-fold less active on guanine than wild-type enzyme: The net change in specificity is 2.5e6-fold. The structure of the designed protein was confirmed by X-ray crystallographic analysis: The remodeled loop adopts a conformation that is within 1-A Calpha RMSD of the computational model.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{276,

title = {Comparative analysis of mutant tyrosine kinase chemical rescue},

author = { Kathryn E Muratore and Markus A Seeliger and Zhihong Wang and Dina Fomina and Johnathan Neiswinger and James J Havranek and David Baker and John Kuriyan and Philip A Cole},

issn = {1520-4995},

year  = {2009},

date = {2009-04-01},

journal = {Biochemistry},

volume = {48},

pages = {3378-86},

abstract = {Protein tyrosine kinases are critical cell signaling enzymes. These enzymes have a highly conserved Arg residue in their catalytic loop which is present two residues or four residues downstream from an absolutely conserved Asp catalytic base. Prior studies on protein tyrosine kinases Csk and Src revealed the potential for chemical rescue of catalytically deficient mutant kinases (Arg to Ala mutations) by small diamino compounds, particularly imidazole; however, the potency and efficiency of rescue was greater for Src. This current study further examines the structural and kinetic basis of rescue for mutant Src as compared to mutant Abl tyrosine kinase. An X-ray crystal structure of R388A Src revealed the surprising finding that a histidine residue of the N-terminus of a symmetry-related kinase inserts into the active site of the adjacent Src and mimics the hydrogen-bonding pattern seen in wild-type protein tyrosine kinases. Abl R367A shows potent and efficient rescue more comparable to Src, even though its catalytic loop is more like that of Csk. Various enzyme redesigns of the active sites indicate that the degree and specificity of rescue are somewhat flexible, but the overall properties of the enzymes and rescue agents play an overarching role. The newly discovered rescue agent 2-aminoimidazole is about as efficient as imidazole in rescuing R/A Src and Abl. Rate vs pH studies with these imidazole analogues suggest that the protonated imidazolium is the preferred form for chemical rescue, consistent with structural models. The efficient rescue seen with mutant Abl points to the potential of this approach to be used effectively to analyze Abl phosphorylation pathways in cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{275,

title = {A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450(BM3)},

author = { Jeffrey A Dietrich and Yasuo Yoshikuni and Karl J Fisher and Frank X Woolard and Denise Ockey and Derek J McPhee and Neil S Renninger and Michelle C Y Chang and David Baker and Jay D Keasling},

issn = {1554-8937},

year  = {2009},

date = {2009-04-01},

journal = {ACS chemical biology},

volume = {4},

pages = {261-7},

abstract = {Production of fine chemicals from heterologous pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved, and the enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450(BM3) from Bacillus megaterium. Using a computer model, we illustrate how key P450(BM3) active site mutations enable binding of the non-native substrate amorphadiene. Incorporating these mutations into P450(BM3) enabled the selective oxidation of amorphadiene artemisinic-11S,12-epoxide, at titers of 250 mg L(-1) in E. coli. We also demonstrate high-yielding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high-value antimalarial drug artemisinin.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{145,

title = {Catalytic mechanism and performance of computationally designed enzymes for Kemp elimination},

author = { Anastassia N Alexandrova and Daniela R"othlisberger and David Baker and William L Jorgensen},

issn = {1520-5126},

year  = {2008},

date = {2008-11-01},

journal = {Journal of the American Chemical Society},

volume = {130},

pages = {15907-15},

abstract = {A series of enzymes for Kemp elimination of 5-nitrobenzisoxazole has been recently designed and tested. In conjunction with the design process, extensive computational analyses were carried out to evaluate the potential performance of four of the designs, as presented here. The enzyme-catalyzed reactions were modeled using mixed quantum and molecular mechanics (QM/MM) calculations in the context of Monte Carlo (MC) statistical mechanics simulations. Free-energy perturbation (FEP) calculations were used to characterize the free-energy surfaces for the catalyzed reactions as well as for reference processes in water. The simulations yielded detailed information about the catalytic mechanisms, activation barriers, and structural evolution of the active sites over the course of the reactions. The catalytic mechanism for the designed enzymes KE07, KE10(V131N), and KE15 was found to be concerted with proton transfer, generally more advanced in the transition state than breaking of the isoxazolyl N-O bond. On the basis of the free-energy results, all three enzymes were anticipated to be active. Ideas for further improvement of the enzyme designs also emerged. On the technical side, the synergy of parallel QM/MM and experimental efforts in the design of artificial enzymes is well illustrated.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{147,

title = {On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes},

author = { Hyundae D Cho and Vanita D Sood and David Baker and Alan M Weiner},

issn = {1469-9001},

year  = {2008},

date = {2008-07-01},

journal = {RNA},

volume = {14},

pages = {1284-9},

abstract = {Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3textquoteright-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{230,

title = {Kemp elimination catalysts by computational enzyme design},

author = { Daniela R"othlisberger and Olga Khersonsky and Andrew M Wollacott and Lin Jiang and Jason DeChancie and Jamie Betker and Jasmine L Gallaher and Eric A Althoff and Alexandre Zanghellini and Orly Dym and Shira Albeck and Kendall N Houk and Dan S Tawfik and David Baker},

issn = {1476-4687},

year  = {2008},

date = {2008-05-01},

journal = {Nature},

volume = {453},

pages = {190-5},

abstract = {The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{151,

title = {De novo computational design of retro-aldol enzymes},

author = { Lin Jiang and Eric A Althoff and Fernando R Clemente and Lindsey Doyle and Daniela R"othlisberger and Alexandre Zanghellini and Jasmine L Gallaher and Jamie L Betker and Fujie Tanaka and Carlos F Barbas and Donald Hilvert and Kendall N Houk and Barry L Stoddard and David Baker},

issn = {1095-9203},

year  = {2008},

date = {2008-03-01},

journal = {Science},

volume = {319},

pages = {1387-91},

abstract = {The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{166,

title = {New algorithms and an in silico benchmark for computational enzyme design},

author = { Alexandre Zanghellini and Lin Jiang and Andrew M Wollacott and Gong Cheng and Jens Meiler and Eric A Althoff and Daniela R"othlisberger and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/07/zanghellini06A.pdf},

issn = {0961-8368},

year  = {2006},

date = {2006-12-01},

journal = {Protein science : a publication of the Protein Society},

volume = {15},

pages = {2785-94},

abstract = {The creation of novel enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Here we describe two new algorithms for enzyme design that employ hashing techniques to allow searching through large numbers of protein scaffolds for optimal catalytic site placement. We also describe an in silico benchmark, based on the recapitulation of the active sites of native enzymes, that allows rapid evaluation and testing of enzyme design methodologies. In the benchmark test, which consists of designing sites for each of 10 different chemical reactions in backbone scaffolds derived from 10 enzymes catalyzing the reactions, the new methods succeed in identifying the native site in the native scaffold and ranking it within the top five designs for six of the 10 reactions. The new methods can be directly applied to the design of new enzymes, and the benchmark provides a powerful in silico test for guiding improvements in computational enzyme design.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{152,

title = {Computational redesign of endonuclease DNA binding and cleavage specificity},

author = { Justin Ashworth and James J Havranek and Carlos M Duarte and Django Sussman and Raymond J Monnat and Barry L Stoddard and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/08/ashworth06A.pdf},

issn = {1476-4687},

year  = {2006},

date = {2006-06-01},

journal = {Nature},

volume = {441},

pages = {656-9},

abstract = {The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site approximately 10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{299,

title = {A model of anthrax toxin lethal factor bound to protective antigen},

author = { D Borden Lacy and Henry C Lin and Roman A Melnyk and Ora Schueler-Furman and Laura Reither and Kristina Cunningham and David Baker and R John Collier},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/lacy05A.pdf},

issn = {0027-8424},

year  = {2005},

date = {2005-11-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {102},

pages = {16409-14},

abstract = {Anthrax toxin is made up of three proteins: the edema factor (EF), lethal factor (LF) enzymes, and the multifunctional protective antigen (PA). Proteolytically activated PA heptamerizes, binds the EF/LF enzymes, and forms a pore that allows for EF/LF passage into host cells. Using directed mutagenesis, we identified three LF-PA contact points defined by a specific disulfide crosslink and two pairs of complementary charge-reversal mutations. These contact points were consistent with the lowest energy LF-PA complex found by using Rosetta protein-protein docking. These results illustrate how biochemical and computational methods can be combined to produce reliable models of large complexes. The model shows that EF and LF bind through a highly electrostatic interface, with their flexible N-terminal region positioned at the entrance of the heptameric PA pore and thus poised to initiate translocation in an N- to C-terminal direction.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{298,

title = {Computational thermostabilization of an enzyme},

author = { Aaron Korkegian and Margaret E Black and David Baker and Barry L Stoddard},

url = {https://www.bakerlab.org/wp-content/uploads/2016/07/korkegian05A.pdf},

issn = {1095-9203},

year  = {2005},

date = {2005-05-01},

journal = {Science},

volume = {308},

pages = {857-60},

abstract = {Thermostabilizing an enzyme while maintaining its activity for industrial or biomedical applications can be difficult with traditional selection methods. We describe a rapid computational approach that identified three mutations within a model enzyme that produced a 10 degrees C increase in apparent melting temperature T(m) and a 30-fold increase in half-life at 50 degrees C, with no reduction in catalytic efficiency. The effects of the mutations were synergistic, giving an increase in excess of the sum of their individual effects. The redesigned enzyme induced an increased, temperature-dependent bacterial growth rate under conditions that required its activity, thereby coupling molecular and metabolic engineering.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{185,

title = {Design, activity, and structure of a highly specific artificial endonuclease},

author = { Brett S Chevalier and Tanja Kortemme and Meggen S Chadsey and David Baker and Raymond J Monnat and Barry L Stoddard},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/chevalier02A.pdf},

issn = {1097-2765},

year  = {2002},

date = {2002-10-01},

journal = {Molecular cell},

volume = {10},

pages = {895-905},

abstract = {We have generated an artificial highly specific endonuclease by fusing domains of homing endonucleases I-DmoI and I-CreI and creating a new 1400 A(2) protein interface between these domains. Protein engineering was accomplished by combining computational redesign and an in vivo protein-folding screen. The resulting enzyme, E-DreI (Engineered I-DmoI/I-CreI), binds a long chimeric DNA target site with nanomolar affinity, cleaving it precisely at a rate equivalent to its natural parents. The structure of an E-DreI/DNA complex demonstrates the accuracy of the protein interface redesign algorithm and reveals how catalytic function is maintained during the creation of the new endonuclease. These results indicate that it may be possible to generate novel highly specific DNA binding proteins from homing endonucleases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{570,

title = {The role of pro regions in protein folding.},

author = { D Baker and A K Shiau and D A Agard},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/theroleofproregions_Baker1993.pdf},

issn = {0955-0674},

year  = {1993},

date = {1993-12-01},

journal = {Current Opinion in Cell Biology},

volume = {5},

pages = {966-70},

abstract = {In vivo, many proteases are synthesized as larger precursors. During the maturation process, the catalytically active protease domain is released from the larger polypeptide or pro-enzyme by one or more proteolytic processing steps. In several well studied cases, amino-terminal pro regions have been shown to play a fundamental role in the folding of the associated protease domains. The mechanism by which pro regions facilitate folding appears to be significantly different from that used by the molecular chaperones. Recent results suggest that the pro region assisted folding mechanism may be used by a wide variety of proteases, and perhaps even by non-proteases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{328,

title = {Protease pro region required for folding is a potent inhibitor of the mature enzyme},

author = { D Baker and J L Silen and D A Agard},

issn = {0887-3585},

year  = {1992},

date = {1992-04-01},

journal = {Proteins},

volume = {12},

pages = {339-44},

abstract = {alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}