Structural dynamics of the protein translocase motor

More than a third of cellular proteins are secreted from the cytoplasm, where they are synthesized, across membranes. This essential cellular process typically engages membrane-embedded channels like the ubiquitous and essential Sec translocase. Secreted proteins and the translocase assemble in large complexes and demonstrate remarkable, multi-level dynamics. These are unapproachable with crystallography and NMR. Instead, this proposal will determine the structural dynamics of the translocase motor SecA and its channel SecYEG by combining enzymology with biophysical state-of-the-art tools: solution-based and immobilized smFRET, local HDX-MS and MD simulation. Special focus will be placed on the preprotein binding domain (PBD) motions of SecA and their link to the protein quaternary dynamics and subsequently to translocation work. I will characterize these states and define the oligomeric state and internal allosteric switches in SecA that control conformational flow. Next, I will monitor any changes in PBD dynamics and SecA’s dimer to monomer quaternary dynamics, upon addition of its physiological ligands and it undergoes the protein receptor to motor transition. Modification of its intrinsic elements will provide tools for freezing and manipulating these states. If and how PBD motions are directly coupled to translocation work will be directly tested by first examining their effect on SecYEG structural dynamics and then by determining their interactions with the chain