337,090 research outputs found

    Unfolding simulations reveal the mechanism of extreme unfolding cooperativity in the kinetically stable alpha-lytic protease.

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    Kinetically stable proteins, those whose stability is derived from their slow unfolding kinetics and not thermodynamics, are examples of evolution's best attempts at suppressing unfolding. Especially in highly proteolytic environments, both partially and fully unfolded proteins face potential inactivation through degradation and/or aggregation, hence, slowing unfolding can greatly extend a protein's functional lifetime. The prokaryotic serine protease alpha-lytic protease (alphaLP) has done just that, as its unfolding is both very slow (t(1/2) approximately 1 year) and so cooperative that partial unfolding is negligible, providing a functional advantage over its thermodynamically stable homologs, such as trypsin. Previous studies have identified regions of the domain interface as critical to alphaLP unfolding, though a complete description of the unfolding pathway is missing. In order to identify the alphaLP unfolding pathway and the mechanism for its extreme cooperativity, we performed high temperature molecular dynamics unfolding simulations of both alphaLP and trypsin. The simulated alphaLP unfolding pathway produces a robust transition state ensemble consistent with prior biochemical experiments and clearly shows that unfolding proceeds through a preferential disruption of the domain interface. Through a novel method of calculating unfolding cooperativity, we show that alphaLP unfolds extremely cooperatively while trypsin unfolds gradually. Finally, by examining the behavior of both domain interfaces, we propose a model for the differential unfolding cooperativity of alphaLP and trypsin involving three key regions that differ between the kinetically stable and thermodynamically stable classes of serine proteases

    Stretching Single Domain Proteins: Phase Diagram and Kinetics of Force-Induced Unfolding

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    Single molecule force spectroscopy reveals unfolding of domains in titin upon stretching. We provide a theoretical framework for these experiments by computing the phase diagrams for force-induced unfolding of single domain proteins using lattice models. The results show that two-state folders (at zero force) unravel cooperatively whereas stretching of non-two-state folders occurs through intermediates. The stretching rates of individual molecules show great variations reflecting the heterogeneity of force-induced unfolding pathways. The approach to the stretched state occurs in a step-wise "quantized" manner. Unfolding dynamics depends sensitively on topology. The unfolding rates increase exponentially with force f till an optimum value which is determined by the barrier to unfolding when f=0. A mapping of these results to proteins shows qualitative agreement with force-induced unfolding of Ig-like domains in titin. We show that single molecule force spectroscopy can be used to map the folding free energy landscape of proteins in the absence of denaturants.Comment: 12 pages, Latex, 6 ps figure

    Direction dependent mechanical unfolding and Green Fluorescent Protein as a force sensor

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    An Ising--like model of proteins is used to investigate the mechanical unfolding of the Green Fluorescent Protein along different directions. When the protein is pulled from its ends, we recover the major and minor unfolding pathways observed in experiments. Upon varying the pulling direction, we find the correct order of magnitude and ranking of the unfolding forces. Exploiting the direction dependence of the unfolding force at equilibrium, we propose a force sensor whose luminescence depends on the applied force.Comment: to appear in Phys Rev

    Mechanical Unfolding of a Simple Model Protein Goes Beyond the Reach of One-Dimensional Descriptions

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    We study the mechanical unfolding of a simple model protein. The Langevin dynamics results are analyzed using Markov-model methods which allow to describe completely the configurational space of the system. Using transition path theory we also provide a quantitative description of the unfolding pathways followed by the system. Our study shows a complex dynamical scenario. In particular, we see that the usual one-dimensional picture: free-energy vs end-to-end distance representation, gives a misleading description of the process. Unfolding can occur following different pathways and configurations which seem to play a central role in one-dimensional pictures are not the intermediate states of the unfolding dynamics.Comment: 10 pages, 6 figure
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