Regulated Unfolding in Electric Fields: Implications for Protein Translocation across Mitochondrial Membranes Ioan Andricioaei, University of Michigan Simulated difference between spontaneous vs. regulated unfolding; the latter is important upon protein import into mitochondria through membrane pores. Simulation of the unfolding of cytochrome b2-fused barnase include effect of membrane external field E(x,t) and repetitive pulling through pore (see Figure). - time-scale problems solved by a novel force extrapolation; procedure based on stochastic path integrals. Extended simulation of large scale conformational changes to RNA molecules. In the long run: From a nanotechnology point of view, nanostructured surface could impose various structural constraint to adsorbed proteins, surface potential, flow can impose forces. Understanding protein folding/unfolding under such diverse conditions present both great challenges and unprecedented opportunitie. We plan to simulate external electric fields for biomolecules immobilized on dielectric or metallic surfaces, such as the DNA chips. Also started to study effect of circular amino-acid permutations (topological changes) on the import mechanism/rates.  Although biologically active while folded, many proteins, such as those translocated through membranes or those degraded by ATP-dependent proteases, must undergo regulated unfolding. Precursor proteins, synthesized in the cytoplasm, are imported inside the mitochondria through a proteinaceous pore. In order to pass them through the import pore, mitochondria unfold precursors by unraveling them from their N-termini. Some precursors have targeting sequences long enough to span the mitochondrial membrane and reach the inner protein import machine, mitochondrial Hsp70, which unfolds them using ATP hydrolysis. For other precursor proteins, with a targeting sequence too short to achieve such a span, an intriguing unfolding mechanism has been suggested, based on measurements of the import rate at different chemical conditions. It has been proposed that many precursors are unfolded by the electrical potential at the mitochondrial membrane. In a short project, we will address this particular aspect of protein import. Using a modified force-field in which each atom feels an additional external force proportional to its charge, we will simulate the unfolding of the cytochrmome b2-fused barnase precursors studied experimentally. Simulations will be performed with and without the external electric field in order to deconvolute the effect of the electrical potential gradient. Time-scale problems due to the presence of energy barriers can be alleviated by a potential smoothing algorithm that preserves the location of the minima. Possibly, several issues related to polarization, like the effect of the electrical moments induced by the external field on the unfolding behavior, could be addressed. Noting that previous observations indicate faster unfolding for protein subdomains than for the overall structure, we will monitor the degree of partial unfolding, and compare the spatial extension to the measured dimensions of the import channels. There exists also the possibility to use the formalism of vector Lyapunov function to quantify the stability of the subdomains as they unfold (see Summary of Previous Research). In the long run, we see important insight from simulations in external electric fields for biomolecules imobilized on dielectric or metalic surfaces, such as the DNA chips.