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Changes of protein stiffness during folding detect protein folding intermediates

Journal of Biological Physics volume 40, pages 15–23 (2014)Cite this article

Abstract

Single-molecule force-quench atomic force microscopy (FQ-AFM) is used to detect folding intermediates of a simple protein by detecting changes of molecular stiffness of the protein during its folding process. Those stiffness changes are obtained from shape and peaks of an autocorrelation of fluctuations in end-to-end length of the folding molecule. The results are supported by predictions of the equipartition theorem and agree with existing Langevin dynamics simulations of a simplified model of a protein folding. In the light of the Langevin simulations the experimental data probe an ensemble of random-coiled collapsed states of the protein, which are present both in the force-quench and thermal-quench folding pathways.

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Notes

  1. Typically a polynomial between 7th order (eight terms) and 12th order (13 terms), was fitted to smoothed L vs. time curves. This was done to subtract a fitted L from an actual and fluctuating L. The procedure was to start with the smallest order of a polynomial, and increase it until a fit goodness (measured with a χ 2) would not change by more than several percent.

  2. To be precise, stiffnesses associated with 1st resonance modes of the AFM cantilevers should be referred to. These are at about 10 pN/nm.

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Acknowledgements

The authors acknowledge Prof. Marek Szymoński from Jagellonian University for his help in preparation of this manuscript. This work was supported by the Thierry Johnson Cancer Research Center at KSU, and the National Science Foundation under Award No. EPS-0903806 and matching support from the State of Kansas through Kansas Technology Enterprise Corporation.

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  1. Department of Physics, Kansas State University, Manhattan, KS, 66506-2601, USA

    Katarzyna E. Małek & Robert Szoszkiewicz

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  1. Katarzyna E. Małek
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Correspondence to Robert Szoszkiewicz.

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Małek, K.E., Szoszkiewicz, R. Changes of protein stiffness during folding detect protein folding intermediates. J Biol Phys 40, 15–23 (2014). https://doi.org/10.1007/s10867-013-9331-y

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Keywords

  • STM and AFM manipulations of a single molecule
  • Folding: thermodynamics, statistical mechanics, models, and pathways
  • Mechanical properties of molecules