A Comparative Study of Mechanical Stability of Circular Permutants during Co-Translocational Unfolding of Proteins through Mitochondrial Pore

Abstract

Mitochondrial import machinery catalyses unfolding of the native precursor proteins by trapping some faster local unfolding fluctuations due to specific secondary structural element adjacent to the targeting sequence. On the rupture of the first resistant structure, the rest of the protein unfolds rapidly by cooperative unfolding during import into the mitochondrial matrix. The process of circular permutation on the protein chain can give important information about the connectivity, structure and folding or unfolding kinetics which guides its translocation and subsequent function in the cell. To study the process of co-translocational unfolding and its dependence on the secondary structure at the N-terminus of the protein imported, we conduct a comparative molecular dynamics study of circular permutants of Dihydrofolate reductase(DHFR) using atomistic model in CHARMM. Six Circular Permutants - CP25, CP38, CP78, CP97, CP108, CP133 - are generated such that the new N-terminus leads either to an alpha helix or a beta-strand. using Steered Molecular Dynamics we compute the work distributions for the forced unfolding of each of the CP's and native DHFR using two processes - unfolding through the geometrical constriction of the model pore as in mitochondrial translocation and mechanical unfolding with the C-terminus fixed. In both cases the unfolding force is applied at the N-terminus. A comparison of the free energy profile along the reaction coordinate for each circular permutant can lead to identification of different unfolding pathways and hence import efficiency based on first resistant structure adjacent to the targeting sequence.