Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment
Open accessAbstract
Protein folding is a fundamental process in biology, key to understanding many human diseases. Experimentally, proteins often appear to fold via simple two- or three-state mechanisms involving mainly native-state interactions, yet recent network models built from atomistic simulations of small proteins suggest the existence of many possible metastable states and folding pathways. We reconcile these two pictures in a combined experimental and simulation study of acyl-coenzyme A binding protein (ACBP), a two-state folder (folding time ~10 ms) exhibiting residual unfolded-state structure, and a putative early folding intermediate. Using single-molecule FRET in conjunction with side-chain mutagenesis, we first demonstrate that the denatured state of ACBP at near-zero denaturant is unusually compact and enriched in long-range structure that can be perturbed by discrete hydrophobic core mutations. We then employ ultrafast laminar-flow mixing experiments to study the folding kinetics of ACBP on the microsecond time scale. These studies, along with Trp-Cys quenching measurements of unfolded-state dynamics, suggest that unfolded-state structure forms on a surprisingly slow (~100 μs) time scale, and that sequence mutations strikingly perturb both time-resolved and equilibrium smFRET measurements in a similar way. A Markov state model (MSM) of the ACBP folding reaction, constructed from over 30 ms of molecular dynamics trajectory data, predicts a complex network of metastable stables, residual unfolded-state structure, and kinetics consistent with experiment but no well-defined intermediate preceding the main folding barrier. Taken together, these experimental and simulation results suggest that the previously characterized fast kinetic phase is not due to formation of a barrier-limited intermediate but rather to a more heterogeneous and slow acquisition of unfolded-state structure.
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A combined experimental and computational study of ACBP concluded that a previously-characterized fast kinetic phase did not correspond to population of a specific intermediate structure but was due to heterogeneous structure acquisition within the unfolded state.
“concluding that a previously-characterized fast kinetic phase did not correspond to population of a specific intermediate structure (as had previously been assumed), but was instead due to heterogeneous structure acquisition within the unfolded state [46, 47]”
From Markov state models of biomolecular conformational dynamics (2014) · cites this paper as [47]
MSMs have been applied to protein folding, ligand binding, conformational change, IDP dynamics/kinetics, and ATP permeation.
“Markov state models have been applied to a wide variety of problems, spanning protein folding,402 protein-ligand binding process,403 protein conformational change,404–405 the dynamics and kinetics of IDPs,369,406–407 and ATP permeation through membrane protein channels.408”
From Protein Ensembles: How Does Nature Harness Thermodynamic Fluctuations for Life? The Diverse Functional Roles of Conformational Ensembles in the Cell (2016) · cites this paper as [402]
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- Authors
- Voelz, V. A., Jäger, M., Yao, S., Chen, Y., Zhu, L., Waldauer, S. A., Bowman, G. R., Friedrichs, M., Bakajin, O., Lapidus, L. J., Weiss, S., & Pande, V. S.
- Affiliations
- Stanford University (US)
- University of California, Los Angeles (US)
- Hong Kong University of Science and Technology (HK)
- Michigan State University (US)
- Southeast University (CN)
- University of California, Davis (US)
- California NanoSystems Institute (US)
- Corresponding author
- Weiss, Shimon
- Pande, Vijay S.
- ORCID
- Venue
- Journal of the American Chemical Society
- Publisher
- American Chemical Society (ACS)
- Published
- 2012-07-19
- Volume
- 134
- Issue
- 30
- Pages
- 12565-12577
- ISSN
- 0002-7863 (ISSN-L)
- 1520-5126
- PMC
- PMC3462454
- OpenAlex
- W2129285395
- Cited by
- 140
- References
- 63
- Citations by year
- 2026: 2 · 2025: 2 · 2024: 3 · 2023: 7 · 2022: 5 · 2021: 8 · 2020: 8 · 2019: 7 · 2018: 9 · 2017: 14 · 2016: 13 · 2015: 15 · 2014: 21 · 2013: 18 · 2012: 6
- FWCI
- 7.72
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Journal of the American Chemical Society (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Rare & Special e-Zone (The Hong Kong University of Science and Technology) (submittedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM062868)
- 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
- acyl-CoA binding protein, protein folding, folding intermediates, metastable states, folding kinetics, single-molecule FRET, Markov state models, folding pathways, two-state folding
- MeSH terms
- Animals, Cattle, Markov Chains, Mutagenesis, Site-Directed, Protein Folding, Diazepam Binding Inhibitor, Fluorescence Resonance Energy Transfer, Protein Stability, Molecular Dynamics Simulation, Hydrophobic and Hydrophilic Interactions, Protein Unfolding
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