Designed active-site library reveals thousands of functional GFP variants.

Autor: Weinstein JY; Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel., Martí-Gómez C; Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724, USA., Lipsh-Sokolik R; Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel., Hoch SY; Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel., Liebermann D; Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, 7610001, Israel., Nevo R; Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel., Weissman H; Department of Molecular Chemistry and Materials Science, Weizmann Institute of Science, Rehovot, 7610001, Israel., Petrovich-Kopitman E; Life science Core facilities, Weizmann Institute of Science, Rehovot, 7610001, Israel., Margulies D; Department of Chemical and Structural Biology, Weizmann Institute of Science, Rehovot, 7610001, Israel., Ivankov D; Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia., McCandlish DM; Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724, USA., Fleishman SJ; Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel. sarel@weizmann.ac.il.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2023 May 20; Vol. 14 (1), pp. 2890. Date of Electronic Publication: 2023 May 20.
DOI: 10.1038/s41467-023-38099-z
Abstrakt: 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.
(© 2023. The Author(s).)
Databáze: MEDLINE
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