Designed active-site library reveals thousands of functional GFP variants

Jonathan Yaacov Weinstein, Carlos Martí-Gómez, Rosalie Lipsh-Sokolik, Shlomo Yakir Hoch, Demian Liebermann, Reinat Nevo, Haim Weissman, Ekaterina Petrovich-Kopitman, David Margulies, Dmitry Ivankov, David M. McCandlish and Sarel J. Fleishman ()
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Jonathan Yaacov Weinstein: Weizmann Institute of Science
Carlos Martí-Gómez: Cold Spring Harbor Laboratory
Rosalie Lipsh-Sokolik: Weizmann Institute of Science
Shlomo Yakir Hoch: Weizmann Institute of Science
Demian Liebermann: Weizmann Institute of Science
Reinat Nevo: Weizmann Institute of Science
Haim Weissman: Weizmann Institute of Science
Ekaterina Petrovich-Kopitman: Weizmann Institute of Science
David Margulies: Weizmann Institute of Science
Dmitry Ivankov: Skolkovo Institute of Science and Technology
David M. McCandlish: Cold Spring Harbor Laboratory
Sarel J. Fleishman: Weizmann Institute of Science

Nature Communications, 2023, vol. 14, issue 1, 1-13

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-38099-z

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