ePrints@IIScePrints@IISc Home | About | Browse | Latest Additions | Advanced Search | Contact | Help

Electron Spin Resonance and Fluorescence Studies of the Bound-state Conformation of a Model Protein Substrate to the Chaperone SecB

Panse, Vikram G and Beena, K and Philipp, Reinhard and Trommer, Wolfgang E and Vogel, Pia D and Varadarajan, Raghavan (2001) Electron Spin Resonance and Fluorescence Studies of the Bound-state Conformation of a Model Protein Substrate to the Chaperone SecB. In: Journal of Biological Chemistry, 276 (36). pp. 33681-33688.

[img] PDF
Restricted to Registered users only

Download (253kB) | Request a copy
Official URL: http://www.jbc.org/cgi/reprint/276/36/33681


SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli. We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51. Mutants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of the labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The electron spin resonance data suggest that these cysteine residues are in close proximity (15 Å) when no substrate protein is bound but move away to a distance of greater than 20 Å when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.

Item Type: Journal Article
Publication: Journal of Biological Chemistry
Publisher: American Society for Biochemistry and Molecular Biology
Additional Information: Copyright of this article belongs to American Society for Biochemistry and Molecular Biology
Keywords: Biochemistry;Molecular Biophysics;Cell Biology;Methods Techniques
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited: 28 Sep 2007
Last Modified: 01 Jul 2011 09:11
URI: http://eprints.iisc.ac.in/id/eprint/11977

Actions (login required)

View Item View Item