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 or Sec system is the major route of export for proteins from the cytosol of Escherichia coli and all eubacteria. The Sec system consists of the heterotrimeric translocon SecYEG along with cytosolic factors SecA (the ATPase of the system) and chaperone SecB. Many significant questions remain unanswered regarding SecYEG mediated secretion, including how many SecYEG and SecA copies are required during translocation. My thesis work presents a novel view of Sec system in near native membrane conditions using a single molecule technique known as atomic force microscopy (AFM). AFM is capable of resolving absolute distances down to atomic (~1Å) length scales and can provide real-time, real-space dynamic pictures of macromolecules at work in near-native conditions. Studies on proteoliposomes SecYEG (PLSecYEG) revealed the sidedness and dynamics of SecYEG. The dynamics observed were significant in magnitude (~1 to 10Å) and were attributed to the cytoplasmic loops of SecY. SecA makes large surface area contact to these unstructured loops. These dynamics are reminiscent of the proposed fly-casting mechanism where unstructured loops increase the capture radii of a binding site and hence the boost the binding rate of SecA to SecYEG. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. Next, we imaged SecA engaged on SecYEG and related the structural states observed to the activity of the translocase. Traditional method of adding SecA to preformed PLSecYEG always yielded low activity and AFM measured heights of these complexes were widely distributed with no preferred binding mode of SecA to SecYEG. In contrast, co-assembly of SecA and SecYEG into liposomes increased the translocation activity by fivefold and these complexes had a single preferred height around ~40Å. This activity is similar to vivo assays and the measured topography reveals the importance of integral membrane SecA to the translocon. Lastly we imaged the active Sec system (SecYEG in complex with SecA and ATP or ATP analogs) in the presence of two different precursors. In this work we observed the important dependency of SecA on precursor species. The measured heights of Sec system protrusions with proOmpA were generally in the lower range (~10 to 32 Å) indicating the release of SecA translocation. In contrast, the major height distribution was around ~40-60 Å for pGBP indicating SecA remains bound during translocation. Though this work is ongoing, the oligomeric state of SecA which is present appears to be similar for both precursors. Overall, this work represents the novel view of the topographical details of the general secretory system (Sec system) in near-native conditions.
|School:||University of Missouri - Columbia|
|School Location:||United States -- Missouri|
|Source:||DAI-B 78/03(E), Dissertation Abstracts International|
|Subjects:||Biochemistry, Physics, Biophysics|
|Keywords:||AFM, Membrane, Protein export, SEC system, SecYEG, Single molecule|
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