Designed active-site library reveals thousands of functional GFP variants
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...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-05-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-38099-z |
| _version_ | 1797822780768518144 |
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| author | 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 Sarel J. Fleishman |
| author_facet | 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 Sarel J. Fleishman |
| author_sort | Jonathan Yaacov Weinstein |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-13T10:14:13Z |
| format | Article |
| id | doaj.art-0a700fa200fc4fecb09ddd2b73e61207 |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-13T10:14:13Z |
| publishDate | 2023-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-0a700fa200fc4fecb09ddd2b73e612072023-05-21T11:19:49ZengNature PortfolioNature Communications2041-17232023-05-0114111310.1038/s41467-023-38099-zDesigned active-site library reveals thousands of functional GFP variantsJonathan Yaacov Weinstein0Carlos Martí-Gómez1Rosalie Lipsh-Sokolik2Shlomo Yakir Hoch3Demian Liebermann4Reinat Nevo5Haim Weissman6Ekaterina Petrovich-Kopitman7David Margulies8Dmitry Ivankov9David M. McCandlish10Sarel J. Fleishman11Department of Biomolecular Sciences, Weizmann Institute of ScienceSimons Center for Quantitative Biology, Cold Spring Harbor LaboratoryDepartment of Biomolecular Sciences, Weizmann Institute of ScienceDepartment of Biomolecular Sciences, Weizmann Institute of ScienceDepartment of Chemical and Biological Physics, Weizmann Institute of ScienceDepartment of Biomolecular Sciences, Weizmann Institute of ScienceDepartment of Molecular Chemistry and Materials Science, Weizmann Institute of ScienceLife science Core facilities, Weizmann Institute of ScienceDepartment of Chemical and Structural Biology, Weizmann Institute of ScienceCenter of Life Sciences, Skolkovo Institute of Science and TechnologySimons Center for Quantitative Biology, Cold Spring Harbor LaboratoryDepartment of Biomolecular Sciences, Weizmann Institute of ScienceAbstract 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.https://doi.org/10.1038/s41467-023-38099-z |
| spellingShingle | 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 Sarel J. Fleishman Designed active-site library reveals thousands of functional GFP variants Nature Communications |
| title | Designed active-site library reveals thousands of functional GFP variants |
| title_full | Designed active-site library reveals thousands of functional GFP variants |
| title_fullStr | Designed active-site library reveals thousands of functional GFP variants |
| title_full_unstemmed | Designed active-site library reveals thousands of functional GFP variants |
| title_short | Designed active-site library reveals thousands of functional GFP variants |
| title_sort | designed active site library reveals thousands of functional gfp variants |
| url | https://doi.org/10.1038/s41467-023-38099-z |
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