Computational redesign of endonuclease DNA binding and cleavage specificity Export

@article{ashworth:06, 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.}, author = {Ashworth, Justin and Havranek, James J. and Duarte, Carlos M. and Sussman, Django and Monnat, Raymond J. and Stoddard, Barry L. and Baker, David}, citeulike-article-id = {678819}, citeulike-linkout-0 = {http://dx.doi.org/10.1038/nature04818}, citeulike-linkout-1 = {http://dx.doi.org/10.1038/nature04818}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/16738662}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=16738662}, day = {1}, doi = {10.1038/nature04818}, journal = {Nature}, keywords = {catalysis, design, engineering, enzyme, protein}, month = {June}, number = {7093}, pages = {656--659}, posted-at = {2009-01-08 21:10:19}, priority = {0}, title = {Computational redesign of endonuclease DNA binding and cleavage specificity}, url = {http://dx.doi.org/10.1038/nature04818}, volume = {441}, year = {2006} }

TY - JOUR ID - ashworth:06 L3 - citeulike-article-id:678819 N2 - 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. IS - 7093 JF - Nature EP - 659 TI - Computational redesign of endonuclease DNA binding and cleavage specificity VL - 441 SP - 656 KW - catalysis KW - design KW - engineering KW - enzyme KW - protein AU - Ashworth, Justin AU - Havranek, James AU - Duarte, Carlos AU - Sussman, Django AU - Monnat, Raymond AU - Stoddard, Barry AU - Baker, David PY - 2006/06/01/ UR - http://dx.doi.org/10.1038/nature04818 ER -