Designed active-site library reveals thousands of functional GFP variants
Jonathan J. Weinstein, Carlos Martí‐Gómez, Rosalie Lipsh‐Sokolik, Shlomo Yakir Hoch, Demian Liebermann, Reinat Nevo, Haim Weissman, Ekaterina Petrovich‐Kopitman, David Margulies, Dmitry N. Ivankov, David M. McCandlish, Sarel J. Fleishman
Abstract
Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is sensitive to mutations due to a high density of molecular interactions, substantially reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called high-throughput Functional Libraries (htFuncLib), that designs a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We apply htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recover >16,000 unique designs encoding as many as eight active-site mutations. Many designs exhibit substantial and useful diversity in functional thermostability (up to 96 °C), fluorescence lifetime, and quantum yield. By eliminating incompatible active-site mutations, htFuncLib generates a large diversity of functional sequences. We envision that htFuncLib will be used in one-shot optimization of activity in enzymes, binders, and other proteins.