This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The SecA ATPase is the engine of preprotein translocase, an essential protein translocation system in prokaryotes. SecA interacts with the protein-conducting pore, SecYEG, and uses the energy of ATP to translocate unfolded preproteins across the cytoplasmic membrane. To push the preprotein through SecYEG, SecA undergoes repeated cycles of preprotein binding and release, which are linked to ATP binding and hydrolysis. The goal of the SAXS experiments is to investigate the conformational changes that take place in SecA when preprotein and/or nucleotides are bound.In addition to being an engine for translocation, SecA is a chaperone in the sense that it interacts only with unfolded preprotein, and this has made detailed biophysical studies of the SecA-preprotein interaction impossible because the preprotein will either refold or aggregate during the experiment. To overcome this technical hurdle, we have used immobilized peptide libraries to find short peptide sequences that bind to SecA with high affinity. Using this technology, we have identified a number of 15 residue peptides that are highly soluble and bind to SecA in a nucleotide-dependent manner. We believe that there are significant conformational changes in SecA when it binds these peptides.SecA is a multi-domain, modular protein (Fig. 1 in attached SecA-SAXS.pdf). Based on a variety of structural (Fig. 2) and biochemical data (not shown), we believe the preprotein is bound between the nucleotide binding domains and the preprotein binding domain, requiring conformational changes in SecA (Fig. 3); an example of the attendant changes in SAXS is provided in Fig. 4. Taking advantage of the modular nature of SecA, we have 4 different constructs to use for SAXS experiments: the effect of preprotein and nucleotide binding on the structure of each construct will allow us to map the inter-domain movements that take place and show how ATP binding and hydrolysis are coupled to preprotein binding.
| Orgel, Joseph P R O; Sella, Ido; Madhurapantula, Rama S et al. (2017) Molecular and ultrastructural studies of a fibrillar collagen from octocoral (Cnidaria). J Exp Biol 220:3327-3335 |
| Yazdi, Aliakbar Khalili; Vezina, Grant C; Shilton, Brian H (2017) An alternate mode of oligomerization for E. coli SecA. Sci Rep 7:11747 |
| Sullivan, Brendan; Robison, Gregory; Pushkar, Yulia et al. (2017) Copper accumulation in rodent brain astrocytes: A species difference. J Trace Elem Med Biol 39:6-13 |
| Morris, Martha Clare (2016) Nutrition and risk of dementia: overview and methodological issues. Ann N Y Acad Sci 1367:31-7 |
| Robison, Gregory; Sullivan, Brendan; Cannon, Jason R et al. (2015) Identification of dopaminergic neurons of the substantia nigra pars compacta as a target of manganese accumulation. Metallomics 7:748-55 |
| Gelfand, Paul; Smith, Randy J; Stavitski, Eli et al. (2015) Characterization of Protein Structural Changes in Living Cells Using Time-Lapsed FTIR Imaging. Anal Chem 87:6025-31 |
| Liang, Wenguang G; Ren, Min; Zhao, Fan et al. (2015) Structures of human CCL18, CCL3, and CCL4 reveal molecular determinants for quaternary structures and sensitivity to insulin-degrading enzyme. J Mol Biol 427:1345-1358 |
| Zhou, Hao; Li, Shangyang; Badger, John et al. (2015) Modulation of HIV protease flexibility by the T80N mutation. Proteins 83:1929-39 |
| Kathuria, Sagar V; Kayatekin, Can; Barrea, Raul et al. (2014) Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS. J Mol Biol 426:1980-94 |
| Skinner, John J; Yu, Wookyung; Gichana, Elizabeth K et al. (2014) Benchmarking all-atom simulations using hydrogen exchange. Proc Natl Acad Sci U S A 111:15975-80 |
Showing the most recent 10 out of 100 publications
