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PreprintCited by 352Protein Structure and DynamicsRNA and protein synthesis mechanismsProtein purification and stability

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Mega-scale experimental analysis of protein folding stability in biology and protein design

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Tsuboyama, Kotaro, Dauparas, Justas, Chen, Jonathan, Laine, Elodie, Mohseni Behbahani, Yasser, Weinstein, Jonathan J., Mangan, Niall M., Ovchinnikov, Sergey, Rocklin, Gabriel J.·2022·Nature
DOI 10.1101/2022.12.06.519132PMC PMC10412457

Preprint — published as 10.1038/s41586-023-06328-6 · Paper page

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Abstract

Advances in DNA sequencing and machine learning are illuminating protein sequences and structures on an enormous scale. However, the energetics driving folding are invisible in these structures and remain largely unknown. The hidden thermodynamics of folding can drive disease, shape protein evolution, and guide protein engineering, and new approaches are needed to reveal these thermodynamics for every sequence and structure. We present cDNA display proteolysis, a new method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of ~850,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 354 natural and 188 de novo designed protein domains 40-72 amino acids in length. Using this immense dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate, and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability. One-Sentence Summary: Massively parallel measurement of protein folding stability by cDNA display proteolysis

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Metadata

Authors
Tsuboyama, K., Dauparas, J., Chen, J., Laine, E., Mohseni Behbahani, Y., Weinstein, J. J., Mangan, N. M., Ovchinnikov, S., & Rocklin, G. J.
Affiliations
  • Northwestern University (US)
  • University of Washington (US)
  • Weizmann Institute of Science (IL)
Corresponding author
Rocklin, Gabriel J.
ORCID
Venue
Nature
Publisher
openRxiv
Published
2022-12-06
Volume
620
Issue
7973
Pages
434-444
ISSN
0028-0836 (ISSN-L)
DOI
10.1101/2022.12.06.519132
PMC
PMC10412457
OpenAlex
W4384820649
Cited by
352
References
1
Citations by year
2026: 121 · 2025: 100 · 2024: 99 · 2023: 29 · 2022: 2
FWCI
30.92
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Nature (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by)
Funders
  • Northwestern University
  • Rush University
  • University of Illinois at Urbana-Champaign
  • University of Illinois at Chicago
  • Quest High Performance Computing
  • Japan Society for the Promotion of Science (19J30003)
  • Precursory Research for Embryonic Science and Technology (JPMJPR21E9)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Keywords
protein folding stability, thermodynamic coupling, de novo protein design, protein domains, single amino acid variants, double mutants, protein engineering
MeSH terms
Protein Domains, Amino Acids, Biology, Mutation, Proteins, Thermodynamics, Protein Engineering, Protein Folding, DNA, Complementary, Protein Stability, Proteolysis

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