Abstract Viewer

M. D. Liu*, E. A. Warner*, C. A. Morrissey, C. W. Fick, T. S. Wu, M. Y. Ornelas, G. V. Ochoa, B. Zhang, C. M. Rathbun, W. B. Porterfield, J. A. Prescher, and A. M. Leconte. in press. Statistical Coupling Analysis-guided library design for discovery of mutant luciferases. Biochemistry : . Full Article

Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable, functionally important, and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to the wild type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in unnatural function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library.

Close This Window