Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein
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Proteins attain their function only after folding into a highly organized three-dimensional structure. Much remains to be learned about the mechanisms of folding of large multidomain proteins, which may populate metastable intermediate states on their energy landscapes. Here we introduce a novel method, based on high-throughput single-molecule fluorescence experiments, which is specifically geared towards tracing the dynamics of folding in the presence of a plethora of intermediates. We employ this method to characterize the folding reaction of a three-domain protein, adenylate kinase. Using thousands of single-molecule trajectories and hidden Markov modelling, we identify six metastable states on adenylate kinase's folding landscape. Remarkably, the connectivity of the intermediates depends on denaturant concentration; at low concentration, multiple intersecting folding pathways co-exist. We anticipate that the methodology introduced here will find broad applicability in the study of folding of large proteins, and will provide a more realistic scenario of their conformational dynamics.
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A detailed six-state model of the folding/unfolding kinetics of adeylate kinase was constructed from a large quantity of single-molecule FRET data, permitting the application of transition path theory for the analysis of folding pathways.
“A detailed six-state model of the folding/unfolding kinetics of adeylate kinase was constructed from a large quantity of single-molecule FRET data, permitting the application of transition path theory for the analysis of folding pathways [75].”
From Markov state models of biomolecular conformational dynamics (2014) · cites this paper as [75]
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- Authors
- Pirchi, M., Ziv, G., Riven, I., Cohen, S. S., Zohar, N., Barak, Y., & Haran, G.
- Affiliations
- Weizmann Institute of Science (IL)
- Princeton University (US)
- Corresponding author
- Haran, Gilad
- Venue
- Nature Communications
- Published
- 2011-10-11
- Volume
- 2
- Issue
- 1
- Pages
- 493-493
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC3209527
- OpenAlex
- W2145920789
- Cited by
- 179
- References
- 47
- Citations by year
- 2026: 4 · 2025: 9 · 2024: 5 · 2023: 9 · 2022: 11 · 2021: 10 · 2020: 4 · 2019: 17 · 2018: 16 · 2017: 17 · 2016: 15 · 2015: 21 · 2014: 18 · 2013: 14 · 2012: 9
- FWCI
- 5.33
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Nature Communications (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- Weizmann Institute of Science
- Tel Aviv University
- National Institutes of Health
- National Institute of General Medical Sciences (R01GM080515)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Force Microscopy Techniques and Applications
- Keywords
- protein folding, folding intermediates, metastable states, folding energy landscape, adenylate kinase, multidomain proteins, single-molecule FRET, hidden Markov models, folding pathways, denaturant concentration, conformational dynamics
- MeSH terms
- Algorithms, Markov Chains, Proteins, Spectrometry, Fluorescence, Protein Folding
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