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Diffusion-limited interaction between unfolded polypeptides and the Escherichia coli chaperone SecB
P Fekkes1, T den Blaauwen, A J Driessen
1Department of Microbiology, University of Groningen, Haren, The Netherlands.
Biochemistry
|August 8, 1995
Summary
SecB chaperone protein binds to precursor proteins, facilitating their translocation via interactions with the SecA translocase subunit. This study quantizes the binding kinetics and affinity, revealing rapid association limited by collision rates.
Area of Science:
- Molecular Biology
- Protein Biochemistry
Background:
- SecB acts as a chaperone in Escherichia coli, essential for protein translocation.
- It targets precursor proteins to the SecA subunit of the translocase complex.
Purpose of the Study:
- To investigate the binding characteristics and kinetics of SecB with polypeptide substrates.
- To elucidate the molecular interactions governing SecB-protein targeting.
Main Methods:
- Spectroscopic techniques, including fluorescence spectroscopy.
- Utilized reduced bovine pancreatic trypsin inhibitor (BPTI) as a model substrate.
- Employed acrylodan labeling and stopped-flow kinetics for detailed analysis.
Main Results:
- SecB exhibits high-affinity binding to BPTI (KD = 5.4 nM) in an apolar environment.
- Binding is rapid, with association limited by collision rates (second-order rate constant = 5 x 10^9 M^-1 s^-1).
- Dissociation rate constant was determined to be 48 s^-1.
Conclusions:
- SecB-polypeptide association in vitro is primarily diffusion-limited.
- In vivo, SecB selectively binds nascent polypeptides, with interactions potentially limited by chain elongation rate rather than folding.