The E. coli Sec Reaction Pathway for Cellular Protein Sorting under a Single Molecule Loupe

Abstract

Protein targeting and secretion is an essential biological process in all life forms. In E. coli, the Sec translocase machinery consists of a conserved protein-conducting channel (SecYEG), which associates with cytoplasmic partners such as SecA. Although busily studied, important mechanistic details on bacterial protein targeting and secretion are still lacking. Since protein conformation and dynamics seems to be key to the function of translocases, we therefore carried out quantitative studies on the dynamic conformation of SecA in vitro using single-pair Förster resonance energy transfer (sp-FRET). Here, single-molecule multi-parameter fluorescence detection (MFD) is a useful tool for sp-FRET, and when combined with nanosecond alternating donor/acceptor excitation, it provides a useful and robust readout of both donor and acceptor dye performance. Using FRET restrained structural modeling (FPS), which performs accessible volume calculations, we selected suitable residues for the used FRET pair (Atto488/Alexa647) on SecA. The different positions were mutated to cysteines for the creation of at least 30 different FRET-pairs, and optimal conditions for labeling with maleimide dyes were determined. Until now 7 FRET-pairs, which includes all the different domains, were selected for creating a dynamic 3D model of SecA. At the moment, we are investigating the effect of binding partners (e.g. pre-proteins such as PhoA) and co-factors (e.g. nucleotides, ADP and ATP) which will lead to very detailed SecA dynamics. We already showed that the addition of ADP results in a more stable SecA conformations. Later on we will me move to an in vitro translocation assay to study the mechanism of substrate docking and –translocation on SecYEG, based on functional existing assays which are compatible with single-molecule experiments.