SecA is an essential component for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane the mechanism for this process remains unclear. In this study we identify two specific regions of SecA that may be important for N-terminal membrane interactions. Molecular modeling of SecA from the E. coli and B. subtilus crystal structures, previously determined, revealed that the first 30 amino acids of SecA consists of a helix of amino acids 1-11 connected by a linker (amino acids 12-16) to an amphipathic helix of amino acids 17-30. The first helix is dispensable for SecA activity; however, deletions in the second N-terminal helix, at amino acids 21-25, result in decrease of SecA activity and a deletion of 26 amino acids no longer complements E. coli ts mutant BL21.19. We show that the deletions of N-terminal amino acids that result in the decrease of SecA activity are correlated to the loss of SecA membrane binding and integration in these deletion mutants. In this study we also test the accuracy of a new membrane protein prediction software PSSM_SVM. This program predicted an embedded membrane (EM) region at SecA amino acids 110-118. The predicted sequence represents an unusual prediction for an EM region as most membrane integral regions consist of 15-30 amino acids. Molecular modeling indicated that the region 110-118 is a part of a helix composed of amino acids 107-121 in E. coli SecA, and is indicative of a membrane embedded domain. Site-directed mutagenesis was carried out with several conserved residues, which included L110, L114, and L118 to determine if substitutions at these positions would affect SecA activity. Our data shows that most SecA mutants (including some predicted to be inactive) are active in vivo; however, L110E and L114R mutations rendered the mutated SecA non-functional. All together this study shows that the N-terminal limit of SecA resides at amino acid 26 and that amino acids 21-25 may form a N-terminal membrane binding determinate. Moreover, the predicted EM region may indeed correspond to a functional embedded membrane region for SecA.
| Identifer | oai:union.ndltd.org:GEORGIA/oai:digitalarchive.gsu.edu:biology_diss-1032 |
| Date | 30 November 2007 |
| Creators | Floyd, Jeanetta Holley |
| Publisher | Digital Archive @ GSU |
| Source Sets | Georgia State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Biology Dissertations |
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