Designed active-site library reveals thousands of functional GFP variants

Author:

Weinstein Jonathan YaacovORCID,Martí-Gómez Carlos,Lipsh-Sokolik RosalieORCID,Hoch Shlomo YakirORCID,Liebermann Demian,Nevo Reinat,Weissman Haim,Petrovich-Kopitman Ekaterina,Margulies DavidORCID,Ivankov DmitryORCID,McCandlish David M.,Fleishman Sarel J.ORCID

Abstract

AbstractMutations 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.

Publisher

Springer Science and Business Media LLC

Subject

General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry,Multidisciplinary

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