@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}

}