@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}

}