Structure-Based Derivation of Protein Folding Intermediates and Energies from Optical Tweezers

Aleksander A. Rebane, Lu Ma, Yongli Zhang

Research output: Contribution to journalArticlepeer-review


Optical tweezers (OTs) measure the force-dependent time-resolved extension of a single macromolecule tethered between two trapped beads. From this measurement, it is possible to determine the folding intermediates, energies, and kinetics of the macromolecule. Previous data analysis generally has used the extension as a reaction coordinate to characterize the observed folding transitions. Despite its convenience, the extension poorly describes folding in the absence of force. Here, we chose the contour length of the unfolded polypeptide as a reaction coordinate and modeled the extensions of protein structures along their predicted folding pathways based on high-resolution structures of the proteins in their native states. We included the extension in our model to calculate the total extensions, energies, and transition rates of the proteins as a function of force. We fit these calculations to the corresponding experimental measurements and obtained the best-fit conformations and energies of proteins in different folding states. We applied our method to analyze single-molecule trajectories of two representative protein complexes responsible for membrane fusion, the HIV-1 glycoprotein 41 and the synaptic SNARE proteins, which involved transitions between two and five states, respectively. Nonlinear fitting of the model to the experimental data revealed the structures of folding intermediates and transition states and their associated energies. Our results demonstrate that the contour length is a useful reaction coordinate to characterize protein folding and that intrinsic extensions of protein structures should be taken into account to properly derive the conformations and energies of protein folding intermediates from single-molecule manipulation experiments.

Original languageEnglish (US)
Pages (from-to)441-454
Number of pages14
JournalBiophysical journal
Issue number2
StatePublished - Jan 19 2016

ASJC Scopus subject areas

  • Biophysics


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