Probing the affinity of SecA for signal peptide in different environments.
SecA, the peripheral subunit of the Escherichia coli preprotein translocase, interacts with a number of ligands during export, including signal peptides, membrane phospholipids, and nucleotides. Using fluorescence resonance energy transfer (FRET), we studied the interactions of wild-type (WT) and mutant SecAs with IAEDANS-labeled signal peptide, and how these interactions are modified in the presence of other transport ligands. We find that residues on the third alpha-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and signal peptide. For SecA in aqueous solution, saturation binding data using FRET analysis fit a single-site binding model and yielded a Kd of 2.4 microM. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low signal peptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has two conformational states; our results do not support different oligomeric states of SecA. Using native gel electrophoresis, we establish signal peptide-induced SecA monomerization in both aqueous solution and lipid vesicles. Whereas the affinity of SecA for signal peptide in an aqueous environment is unaffected by temperature or the presence of nucleotides, in lipids the affinity decreases in the presence of ADP or AMP-PCP but increases at higher temperature. The latter finding is consistent with SecA existing in an elongated form while inserting the signal peptide into membranes.
Musial-Siwek
Monika
M
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Rusch
Sharyn L
SL
Kendall
Debra A
DA
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D006358:Hot Temperature; D026901:Membrane Transport Proteins / Q000737:chemistry*; D015335:Molecular Probes; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D015335:Molecular Probes; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins