Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Designed active-site library reveals thousands of functional GFP variants

View ORCID ProfileJonathan Yaacov Weinstein, Carlos Martí-Gómez, View ORCID ProfileRosalie Lipsh-Sokolik, View ORCID ProfileShlomo Yakir Hoch, Demian Liebermann, Reinat Nevo, Haim Weissman, Ekaterina Petrovich-Kopitman, David Margulies, View ORCID ProfileDmitry Ivankov, David McCandlish, View ORCID ProfileSarel Jacob Fleishman
doi: https://doi.org/10.1101/2022.10.11.511732
Jonathan Yaacov Weinstein
1Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Jonathan Yaacov Weinstein
Carlos Martí-Gómez
2Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, 11724, NY, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rosalie Lipsh-Sokolik
1Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Rosalie Lipsh-Sokolik
Shlomo Yakir Hoch
1Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Shlomo Yakir Hoch
Demian Liebermann
3Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot 7610001, Israel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Reinat Nevo
1Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Haim Weissman
4Department of Molecular Chemistry and Materials Science, Weizmann Institute of Science, Rehovot, 7610001 Israel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ekaterina Petrovich-Kopitman
5Life science Core facilities, Weizmann Institute of Science, Rehovot 7610001, Israel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David Margulies
6Department of Chemical and Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dmitry Ivankov
7Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Dmitry Ivankov
David McCandlish
2Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, 11724, NY, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sarel Jacob Fleishman
1Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Sarel Jacob Fleishman
  • For correspondence: sarel.fleishman@weizmann.ac.il
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

Abstract

Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is extremely sensitive to mutations due to a high density of molecular interactions, drastically reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called htFuncLib, to design a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We applied htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recovered >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 useful for one-shot optimization of activity in enzymes, binders, and other proteins.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Response to reviewers.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted January 19, 2023.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Designed active-site library reveals thousands of functional GFP variants
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Designed active-site library reveals thousands of functional GFP variants
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 McCandlish, Sarel Jacob Fleishman
bioRxiv 2022.10.11.511732; doi: https://doi.org/10.1101/2022.10.11.511732
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools
Designed active-site library reveals thousands of functional GFP variants
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 McCandlish, Sarel Jacob Fleishman
bioRxiv 2022.10.11.511732; doi: https://doi.org/10.1101/2022.10.11.511732

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Biochemistry
Subject Areas
All Articles
  • Animal Behavior and Cognition (4237)
  • Biochemistry (9147)
  • Bioengineering (6786)
  • Bioinformatics (24020)
  • Biophysics (12137)
  • Cancer Biology (9544)
  • Cell Biology (13795)
  • Clinical Trials (138)
  • Developmental Biology (7642)
  • Ecology (11715)
  • Epidemiology (2066)
  • Evolutionary Biology (15517)
  • Genetics (10650)
  • Genomics (14332)
  • Immunology (9492)
  • Microbiology (22856)
  • Molecular Biology (9103)
  • Neuroscience (49028)
  • Paleontology (355)
  • Pathology (1484)
  • Pharmacology and Toxicology (2572)
  • Physiology (3848)
  • Plant Biology (8337)
  • Scientific Communication and Education (1472)
  • Synthetic Biology (2296)
  • Systems Biology (6196)
  • Zoology (1302)