The E.coli Sec Pathway 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 Forster resonance energy transfer (FRET) on a home-built multiparameter fluorescence detection microscope with pulsed interleaved excitation (MFD-PIE). Using FRET restrained structural modeling (FPS), we selected suitable FRET pairs on SecA. The different positions were mutated to Cysteines, and optimal conditions for labeling with maleimide dyes were determined. Preliminary FRET measurements were performed that provide insight in the stoichiometry of SecA in solution. The perspective is to study all the SecA mutants to create a dynamic 3D model of SecA. In a second part, we perform a biophysical analysis of the Sec pathway in vivo in E. coli. To do this, we have selected plasmids that allow controllable expression of the proteins. Then, we employed number and brightness analysis and raster image correlation spectroscopy to study the stoichiometry and dynamics of SecA in E. coli using a confocal laser scanning microscope.