Weinstein, Jonathan Yaacov, Martí-Gómez, Carlos, Lipsh-Sokolik, Rosalie, Hoch, Shlomo Yakir, Liebermann, Demian, Nevo, Reinat, Weissman, Haim, Petrovich-Kopitman, Ekaterina, Margulies, David, Ivankov, Dmitry, McCandlish, David M, Fleishman, Sarel J (May 2023) Designed active-site library reveals thousands of functional GFP variants. Nature Communications, 14 (1). p. 2890. ISSN 2041-1723
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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.
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