title: Discriminating between stabilizing and destabilizing protein design mutations via recombination and simulation.
abstract: Accuracy of current computational protein design (CPD) methods is limited by inherent approximations in energy potentials and sampling. These limitations are often used to qualitatively explain design failures; however, relatively few studies provide specific examples or quantitative details that can be used to improve future CPD methods. Expanding the design method to include a library of sequences provides data that is well suited for discriminating between stabilizing and destabilizing design elements. Using thermophilic endoglucanase E1 from Acidothermus cellulolyticus as a model enzyme, we computationally designed a sequence with 60 mutations. The design sequence was rationally divided into structural blocks and recombined with the wild-type sequence. Resulting chimeras were assessed for activity and thermostability. Surprisingly, unlike previous chimera libraries, regression analysis based on one- and two-body effects was not sufficient for predicting chimera stability. Analysis of molecular dynamics simulations proved helpful in distinguishing stabilizing and destabilizing mutations. Reverting to the wild-type amino acid at destabilized sites partially regained design stability, and introducing predicted stabilizing mutations in wild-type E1 significantly enhanced thermostability. The ability to isolate stabilizing and destabilizing elements in computational design offers an opportunity to interpret previous design failures and improve future CPD methods.
authors.affiliation: Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Lucas B
authors.initials: LB
authors.lastname: Johnson
authors.affiliation: Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Lucas P
authors.initials: LP
authors.lastname: Gintner
authors.affiliation: Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Sehoo
authors.initials: S
authors.lastname: Park
authors.affiliation: Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, USA christopher.snow@colostate.edu.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Snow
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D020125:Mutation, Missense; D011995:Recombination, Genetic
chemical_list: D001426:Bacterial Proteins; C056374:endoglucanase 1; D002480:Cellulase
keywords: computational protein design; endoglucanase; molecular dynamics; recombination; thermostability