Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 17, Pages 6963-6968Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1018177108
Keywords
folding intermediates; atomic force microscopy; self-organized polymer model; unfolding kinetics; three stage refolding
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Funding
- National Institutes of Health [GM089685]
- National Science Foundation [CHE 09-14033]
- Klaus Tschira Foundation
- German Academic Exchange Service
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Single molecule experiments that initiate folding using mechanical force are uniquely suited to reveal the nature of populated states in the folding process. Using a strategy proposed on theoretical grounds, which calls for repeated cycling of force from high to low values using force pulses, it was demonstrated in atomic force spectroscopy (AFM) experiments that an ensemble of minimum energy compact structures (MECS) are sampled during the folding of polyubiquitin. The structures in the ensemble are mechanically resistant to a lesser extent than the native state. Remarkably, forced unfolding of the populated intermediates reveals a broad distribution of extensions including steps up to 30 nm and beyond. We show using molecular simulations that favorable interdomain interactions leading to domain swapping between adjacent ubiquitin modules results in the formation of the ensemble of MECS, whose unfolding leads to an unusually broad distribution of steps. We obtained the domain-swapped structures using coarse-grained ubiquitin dimer models by exchanging native interactions between two monomeric ubiquitin molecules. Brownian dynamics force unfolding of the proposed domain-swapped structures, with mechanical stability that is approximately 100-fold lower than the native state, gives rise to a distribution of extensions from 2 to 30 nm. Our results, which are in quantitative agreement with AFM experiments, suggest that domain swapping may be a general mechanism in the assembly of multi-sub-unit proteins.
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