Insights into the molecular mechanisms and dynamics of translocation through SecYEG: an approach using ensemble and single molecule techniques. (360G-Wellcome-080705_Z_06_Z)

1.    Insights into the molecular mechanisms and dynamics of translocation through SecYEG: an approach using ensemble and single molecule techniques Signal sequence-bearing secretory pre-proteins, and integral membrane proteins are translocated either through the inner membrane in bacteria or the endoplasmic reticulum in eukaryotic cells. The main secretory pathway in Escherichia coli involves the SecYEG translocon, which consists of three integral inner membrane proteins: SecY, which forms the protein channel, SecE that forms a clamp around the complex and SecG. A plethora of studies demonstrated that SecYEG undergoes large and complex sequential intramolecular conformational changes upon binding of partner proteins (e.g. SecA or ribosome) and translocation of secretory proteins. However, the nature of the open and closed states, the oligomeric organisation, the regulation of channel gating and the dynamic behaviour of the reaction remain poorly understood. The emergence of fluorescence techniques at the single molecule level has already improved our understanding of such dynamic processes. In-house developments in single molecule fluorescence spectroscopy instrumentation, combined with the expertise of the Radford group in protein folding and the Baldwin group in membrane proteins, place us in a strong position to unravel the SecYEG dynamics and conformational changes involved in protein translocation through this protein pore. The aims of the project are to: (a) Design structure-based FRET labelled mutants of SecYEG, focusing on key domains of the translocon (e.g. "plug domain", "hinge region", dimerisation interface and the two halves of SecYEG, and (b) characterise the structure/function relationship of these labelled SecYEG mutants using ensemble techniques. Measure intra- and intermolecular distances within and between SecYEG and the model substrate proOmpA during translocation using single molecule FRET (smFRET). Determine the extent to which restricting the movement of key domains by "locking" the protein in specific conformations can influence SecYEG function and derive a detailed mechanistic model for protein translocation.