337,090 research outputs found
Unfolding simulations reveal the mechanism of extreme unfolding cooperativity in the kinetically stable alpha-lytic protease.
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
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
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
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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