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

PDF Designed active-site library reveals thousands of functional GFP variants.pdf - Published Version Available under License Creative Commons Attribution. Download (3MB)

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:	McCandlish, David Marti Gomez Aldaravi Carlos
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:	Publisher
URI:	https://repository.cshl.edu/id/eprint/41029

Actions (login required)

Administrator's edit/view item