Structural investigations of the protein export system at the single molecule level
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More than 30% of proteins in any organism are transported from the site of synthesis into or through cell membranes to properly localize and function. The general secretory (Sec system) is the major route of export for proteins from the cytosol of Escherichia coli and all eubacteria. The pathway through the membrane – the translocon – is provided by SecYEG, a protein complex that is highly conserved having homologs across the kingdoms of life. SecA is the ATPase of the Sec system and it binds SecYEG to perform translocation. Recently, atomic force microscopy (AFM) has emerged as an important complementary tool in biophysics and is well suited for studying membrane proteins in near-native conditions. Here we studied purified SecYEG that was reconstituted into liposomes. After confirming activity, changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude and were attributed to the aforementioned loops of SecY. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. We have also imaged SecA engaged on SecYEG and related the structural states observed to the activity of the translocase. Lastly we imaged the active Sec system in the presence of two different precursors. The measured heights of Sec system protrusions with proOmpA were generally in the lower range (~10 to 32 Å) indicating the release of SecA during translocation. In contrast, the major height distribution was around ~40-60 Å for pGBP indicating SecA remains bound during translocation. Overall, this work represents the novel view of the topographical details of the Sec system in near native conditions.