Date of Completion

10-14-2013

Embargo Period

4-12-2014

Major Advisor

DEBRA KENDALL

Associate Advisor

CHARLES GIARDINA

Associate Advisor

VICTORIA ROBINSON

Field of Study

Biochemistry

Open Access

Open Access

Abstract

More than 30% of proteins synthesized in the cytoplasm of cells, must be transported into or across the cell membrane to reach their final destinations. These secretory preproteins are synthesized with an amino-terminal signal peptide, which directs their export via specific protein translocation pathways. SecA is a principal component of this export system in bacterial cells, and functions as an ATPase nanomotor that provides energy via ATP hydrolysis for the translocation of preproteins across a membrane-embedded SecYEG translocon channel. The signal peptide is cleaved following translocation, by a membrane-embedded enzyme known as signal peptidase I (SPase I). Analysis of interactions between the signal peptide, SecA and SPase I is of crucial importance towards understanding the bacterial protein transport process, and could eventually aid in the development of antimicrobial drugs.

In this study, we used multiple biochemical and biophysical approaches to probe interactions between different components of the Sec pathway. Using the substituted cysteine accessibility method (SCAM), we mapped a distinct groove on SecA that binds signal peptides. The accessibility of this binding site is variable, and therefore provides an ideal mechanism for preprotein binding and release. The active oligomeric state and the dimeric interface of SecA are unresolved. We identified a select few residues that lie on the dimer interface of SecA. Our results are consistent with the formation of a parallel dimer. The dimer was found to dissociate upon interaction of SecA with several translocation ligands. These results suggest that monomeric SecA is the translocation-active form of SecA.

Tryptophan fluorescence spectroscopic analysis of a soluble catalytically active form of SPase I (SPase I Δ2-75), indicated that a large hydrophobic region of the periplasmic domain interacts extensively with the membrane, while the rest of the enzyme does not. Studies performed with signal peptide revealed that the peptide positions itself with respect to the membrane and the enzyme, such that the cleavage site is accessible to the enzyme active site. The enzyme also undergoes direct and allosteric structural changes in response to signal peptide binding.

These results provide key insights into fundamental aspects of bacterial protein translocation.

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