Conformational state of the SecYEG-bound SecA probed by single tryptophan fluorescence spectroscopy.
The SecYEG complex is a membrane-embedded channel that permits the passage of precursor proteins (preproteins) across the inner membrane of Escherichia coli. SecA is a molecular motor that associates with the SecYEG pore and drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the conformational state of soluble and SecYEG-bound SecA using single tryptophan mutants of SecA. The fluorescence spectral properties of the single tryptophans of SecA and their accessibility to the quencher acrylamide demonstrate that SecA undergoes a conformational change that results in a more compact structure upon binding of ATP and binding to the SecYEG pore. In addition, SecYEG-bound SecA undergoes ATP-dependent conformational changes that are not observed for soluble SecA. These data support a model in which binding to the SecYEG channel has a major impact on the SecA conformation.
Natale
Paolo
P
Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9750 AA Haren, The Netherlands.
den Blaauwen
Tanneke
T
van der Does
Chris
C
Driessen
Arnold J M
AJ
D020106:Acrylamide / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D021381:Protein Transport / Q000235:genetics; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D012995:Solubility; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C088616:SecG protein, E coli; C057841:SecY protein, E coli; D020106:Acrylamide; D014364:Tryptophan; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins