This project has as its overarching goal elucidation of the roles played by signal sequences in various steps of protein export or integration of proteins into the cytoplasmic membrane. Signal sequences serve as cellular 'zipcodes' in that they carry essentially all of the information necessary to target a protein for export from the cell. Despite considerable progress in our understanding of protein secretion and integration into membranes, the means by which signal sequences on nascent or newly synthesized proteins are specifically recognized, stably bound, and then released at the appropriate time for interaction with membrane components is still poorly understood. The proposed work addresses the question of how these processes are accomplished by the two signal sequence-mediated pathways in E. coli, that involving Ffh, which is homologous to the 54 kDa subunit of the mammalian signal recognition particle (SRP), and that involving SecA, a uniquely prokaryotic protein. To address this goal, we will utilize synthetic peptides corresponding to signal sequences of bacterial proteins as probes. The proposed strategy is to identify the signal sequence-binding sites on Ffh and SecA by cross-linking them to signal peptides; to explore how these binding sites are altered in affinity as the signal sequence is bound and released; and to determine the conformation adopted by a signal sequence bound either to Ffh or to SecA. We hypothesize that both of these proteins have intramolecular sequences that bind to the hydrophobic signal sequence-binding site to protect it in the absence of a nascent or precursor protein. Several diseases (e.g., cystic fibrosis, Alzheimer's, Wolman, and some prion-associated diseases) have been shown to be associated with signal peptide polymorphisms that lead to mislocalization of a secreted protein or to hypersecretion. Elucidation of the mode of recognition, binding, and release of signal sequences by SRP will help efforts to develop therapeutic strategies for these diseases. Additionally, SecA is an ideal target for antibiotics, since it is uniquely present in bacteria. Enhanced understanding of its mode of recognition will provide a basis for development of new antibacterial agents.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biophysical Chemistry Study Section (BBCB)
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Shapiro, Bert I
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University of Massachusetts Amherst
Schools of Arts and Sciences
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Maki, Jenny L; Krishnan, Beena; Gierasch, Lila M (2012) Using a low denaturant model to explore the conformational features of translocation-active SecA. Biochemistry 51:1369-79
Clerico, Eugenia M; Szymanska, Aneta; Gierasch, Lila M (2009) Exploring the interactions between signal sequences and E. coli SRP by two distinct and complementary crosslinking methods. Biopolymers 92:201-11
Clerico, Eugenia M; Maki, Jenny L; Gierasch, Lila M (2008) Use of synthetic signal sequences to explore the protein export machinery. Biopolymers 90:307-19
Krishnan, Beena; Gierasch, Lila M (2008) Cross-strand split tetra-Cys motifs as structure sensors in a beta-sheet protein. Chem Biol 15:1104-15
Krishnan, Beena; Szymanska, Aneta; Gierasch, Lila M (2007) Site-specific fluorescent labeling of poly-histidine sequences using a metal-chelating cysteine. Chem Biol Drug Des 69:31-40
Mainprize, Iain L; Beniac, Daniel R; Falkovskaia, Elena et al. (2006) The structure of Escherichia coli signal recognition particle revealed by scanning transmission electron microscopy. Mol Biol Cell 17:5063-74
Lin, Bor-Ruei; Gierasch, Lila M; Jiang, Chun et al. (2006) Electrophysiological studies in Xenopus oocytes for the opening of Escherichia coli SecA-dependent protein-conducting channels. J Membr Biol 214:103-13
Swain, Joanna F; Gierasch, Lila M (2005) First glimpses of a chaperonin-bound folding intermediate. Proc Natl Acad Sci U S A 102:13715-6
Chou, Yi-Te; Gierasch, Lila M (2005) The conformation of a signal peptide bound by Escherichia coli preprotein translocase SecA. J Biol Chem 280:32753-60
Fak, John J; Itkin, Anna; Ciobanu, Daita D et al. (2004) Nucleotide exchange from the high-affinity ATP-binding site in SecA is the rate-limiting step in the ATPase cycle of the soluble enzyme and occurs through a specialized conformational state. Biochemistry 43:7307-27

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