@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}

}