Diffusion-limited interaction between unfolded polypeptides and the Escherichia coli chaperone SecB.
SecB is a chaperone dedicated to protein translocation in Escherichia coli. SecB binds to a subset of precursor proteins, and targets them in a translocation-competent state to the SecA subunit of the translocase. The nature and kinetics of the interaction of SecB with polypeptides were studied by spectroscopic techniques using the reduced form of bovine pancreatic trypsin inhibitor (BPTI) as a model substrate. Binding of SecB to BPTI resulted in an increase in the fluorescence of the surface-exposed tryptophan residue 36 of SecB. SecB reversibly binds BPTI in stoichiometric amounts. Labeling of BPTI with the fluorophore acrylodan allowed the analysis of the binding reaction at nanomolar concentrations. High-affinity binding (KD of 5.4 nM) of labeled BPTI to SecB resulted in a blue shift of the acrylodan emission maximum and an increase in the fluorescence quantum yield, suggesting that BPTI binds in an apolar environment. Stopped-flow acquisition of rate constants of complex formation between SecB and BPTI yielded a second-order binding rate constant of 5 x 10(9) M-1 s-1, and a dissociation rate constant of 48 s-1. These data demonstrate that in vitro, the association of SecB with polypeptide substrates is limited by the rate of collision. In vivo, SecB binding is selective, and predominantly occurs with nascent polypeptides. Since these chains are not expected to fold into stable structures, SecB association may be governed by "more or less" specific interactions and be limited by the rate of chain elongation rather than the rate of folding.
Fekkes
P
P
Department of Microbiology, University of Groningen, Haren, The Netherlands.
den Blaauwen
T
T
Driessen
A J
AJ
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D007611:Aprotinin / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004058:Diffusion; D017022:Flow Injection Analysis; D005456:Fluorescent Dyes; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C057842:SecB protein, Bacteria; C038518:acrylodan; D014364:Tryptophan; D007611:Aprotinin; D015081:2-Naphthylamine