SecA folding kinetics: a large dimeric protein rapidly forms multiple native states.
SecA, a 202 kDa dimeric protein, is the ATPase for the Sec-dependent translocase of precursor proteins in vivo. SecA must undergo conformational changes, which may involve dissociation into a monomer, as it translocates the precursor protein across the inner membrane. To better understand the dynamics of SecA in vivo, protein folding studies to probe the native, intermediate, and unfolded species of SecA in vitro have been done. SecA folds through a stable dimeric intermediate and dimerizes in the dead-time of a manual-mixing kinetic experiment ( approximately 5-7 seconds). Here, stopped-flow fluorescence and CD, as well as ultra-rapid continuous flow fluorescence techniques, were used to further probe the rapid folding kinetics of SecA. In the absence of urea, rapid, near diffusion-limited ( approximately 10(9)M(-1)s(-1)) SecA dimerization occurs following a rate-limiting unimolecular rearrangement of a rapidly formed intermediate. Multiple kinetic folding and unfolding phases were observed and SecA was shown to have multiple native and unfolded states. Using sequential-mixing stopped-flow experiments, SecA was determined to fold via parallel channels with sequential intermediates. These results confirm that SecA is a highly dynamic protein, consistent with the rapid, major conformational changes it must undergo in vivo.
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, USA.
Shannon M
SM
Doyle
Osman
O
Bilsel
Carolyn M
CM
Teschke
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019281:Dimerization; D007700:Kinetics; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017510:Protein Folding; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013997:Time Factors; D014508:Urea / Q000378:metabolism
D001426:Bacterial Proteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D014508:Urea; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins