Article

Nonkinetic Modeling of the Mechanical Unfolding of Multimodular Proteins: Theory and Experiments

Laboratory of Physics of Living Matter, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Biophysical Journal (Impact Factor: 3.83). 09/2011; 101(6):1504-12. DOI: 10.1016/j.bpj.2011.07.047
Source: PubMed

ABSTRACT We introduce and discuss a novel approach called back-calculation for analyzing force spectroscopy experiments on multimodular proteins. The relationship between the histograms of the unfolding forces for different peaks, corresponding to a different number of not-yet-unfolded protein modules, is exploited in such a manner that the sole distribution of the forces for one unfolding peak can be used to predict the unfolding forces for other peaks. The scheme is based on a bootstrap prediction method and does not rely on any specific kinetic model for multimodular unfolding. It is tested and validated in both theoretical/computational contexts (based on stochastic simulations) and atomic force microscopy experiments on (GB1)(8) multimodular protein constructs. The prediction accuracy is so high that the predicted average unfolding forces corresponding to each peak for the GB1 construct are within only 5 pN of the averaged directly-measured values. Experimental data are also used to illustrate how the limitations of standard kinetic models can be aptly circumvented by the proposed approach.

0 Followers
 · 
162 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an AFM cantilever and a glass surface using HaloTag anchoring at one end, and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ~2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a four-fold increase, from 131 pN to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice more sensitive to pulling force compared to protein L, and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.
    Journal of the American Chemical Society 08/2013; 135(34). DOI:10.1021/ja4056382 · 11.44 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Bell–Evans model which predicts the linear dependence of the most probable intermolecular bond rupture force on the logarithm of stretching force loading rate is usually used to discuss the dynamic force spectroscopy experiment data. This model is consistent with the Kramers' theory of the bond dissociation rate only if one presupposes an independence of the pre-exponential factors in the Kramers relation on the acting force and a linear decrease of the dissociation barrier height on this same force, and for this to be true rather special shape of the interaction landscape is required. Here, we present a first order correction to this model (first terms of corresponding Taylor expansions are taken into account), discuss its implication for the interpretation of dynamic force spectroscopy experiment data and compare our model with the Monte Carlo simulation of a specially designed single molecule dynamic force spectroscopy experiment. In addition to the most probable bond rupture force, an average rupture force values are also calculated. All approximations made and the range of applicability of the obtained results are carefully described and compared with those for some other models in the field.
    Journal of Applied Physics 07/2013; 114(3). DOI:10.1063/1.4815869 · 2.19 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Heterogeneity in biological molecules, resulting in molecule-to-molecule variations in their dynamics and function, is an emerging theme. To elucidate the consequences of heterogeneous behavior at the single molecule level, we propose an exactly solvable model in which the unfolding rate due to mechanical force depends parametrically on an auxiliary variable representing an entropy barrier arising from fluctuations in internal dynamics. When the rate of fluctuations-a measure of dynamical disorder-is comparable to or smaller than the rate of force-induced unbinding, we show that there are two experimentally observable consequences: nonexponential survival probability at constant force, and a heavy-tailed rupture force distribution at constant loading rate. By fitting our analytical expressions to data from single molecule pulling experiments on proteins and DNA, we quantify the extent of disorder. We show that only by analyzing data over a wide range of forces and loading rates can the role of disorder due to internal dynamics be quantitatively assessed.
    Physical Review Letters 04/2014; 112(13):138101. DOI:10.1103/PhysRevLett.112.138101 · 7.73 Impact Factor

Full-text (2 Sources)

Download
42 Downloads
Available from
May 22, 2014