Designed active-site library reveals thousands of functional GFP variants

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.

Item Type: Paper
Subjects: bioinformatics
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
bioinformatics > computational biology > algorithms
bioinformatics > computational biology
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > green fluorescent protein
bioinformatics > computational biology > algorithms > machine learning
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > mutations
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types
CSHL Authors:
Communities: CSHL labs > McCandlish lab
SWORD Depositor: CSHL Elements
Depositing User: CSHL Elements
Date: 20 May 2023
Date Deposited: 28 Sep 2023 17:22
Last Modified: 11 Jan 2024 14:46
PMCID: PMC10199939
Related URLs:
URI: https://repository.cshl.edu/id/eprint/41029

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