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42371-G7
Regulated Unfolding in Electric Fields: Implications for Protein Translocation across Mitochondrial Membranes
Funding from this grant enabled us to continue work in the area of the effect of forces on the large scale conformational transitions that proteins undergo upon application of force (as happens when proteins undergo passage through pores). We have established a collaboration with Dr Liviu Movileanu, Syracuse University and have obtained results (paper in progress) on the alpha hemolysin pore. Our simulation results are put in the context of single-molecule intensity-current curves Dr Movileanu is measuring.
Trajectories of unfolding proteins on which circular permutations have beeen done have also been accumulated (work in progress) and partial free energy profiles along the unfolding coordinate upon passage through pores have been calculated.
In two published papers this year, work supported from the ACS-PRF grant has enabled us to extend the results into two new exciting directions.
Firstly, we have developed a rigorous procedure that enabled us to do an exact low force extrapolation from simulations or experiments at high values of force. This was needed in the context of the work supported by ACS_PRF because mechanical forces play a key role in regulated unfolding in the cell, as well as in novel single-molecule pulling experiments. We presented an exact method that enables one to extrapolate, to low ( or zero) forces, entire time-correlation functions and kinetic rate constants from the conformational dynamics either simulated numerically or measured experimentally at a single, relatively higher, external force. The method has twofold relevance: 1), to extrapolate the kinetics at physiological force conditions from molecular dynamics trajectories generated at higher forces that accelerate conformational transitions; and 2), to extrapolate unfolding rates from experimental force-extension single-molecule curves. The theoretical formalism, based on stochastic path integral weights of Langevin trajectories, was presented for the constant-force, constant loading rate, and constant-velocity modes of the pulling experiments.
Secondly, extending our interest in the large scale unfolding changes suffered by proteins upon import, we have also looked at the large scale conformational changes in RNA molecules. As such, we have studied an HIV TAR RNA element, in collaboration with experimentalist colleague, Prof. Hashim Al-Hashimi (Univ of Michigan-Biophysics). To deal with the significant coupling between overall diffusional rotation and large scale conformational motion in TAR, we utilized a novel isotropic reorientational eigenmode dynamics analysis of simulated molecular trajectories to obtain a detailed description of TAR dynamics and an accurately quantified pattern of dynamical correlations. The analysis demonstrated inseparability of conformational and overall motional modes, and confirmed the existence of collective dynamics.
In the future, the work supported by ACS-PRF will be a good experience in our interest to simulate unfolding of proteins involved in the proteasome, another exciting case of regulated unfolding.
