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Guanidine hydrochloride unfolding of a transmembrane beta-strand in FepA using site-directed spin labeling. Protein Sci 1998 Jun;7(6):1469-76

Date

07/09/1998

Pubmed ID

9655352

Pubmed Central ID

PMC2144043

DOI

10.1002/pro.5560070624

Scopus ID

2-s2.0-0031777549 (requires institutional sign-in at Scopus site)   33 Citations

Abstract

We have used the electron spin resonance (ESR) site-directed spin-labeling (SDSL) technique to examine the guanidine hydrochloride (Gdn-HCl) induced denaturation of several sites along a transmembrane beta-strand located in the ferric enterobactin receptor, FepA. In addition, we have continued the characterization of the beta-strand previously identified by our group (Klug CS et al., 1997, Biochemistry 36:13027-13033) to extend from the periplasm to the extracellular surface loop in FepA, an integral membrane protein containing a beta-barrel motif comprised of a series of antiparallel beta-strands that is responsible for transport of the iron chelate, ferric enterobactin (FeEnt), across the outer membrane of Escherichia coli and many related enteric bacteria. We have previously shown that a large surface loop in FepA containing the FeEnt binding site denatures independently of the beta-barrel domain (Klug CS et al., 1995, Biochemistry 34:14230-14236). The SDSL approach allows examination of the unfolding at individual residues independent of the global unfolding of the protein. This work shows that sites along the beta-strand that are exposed to the aqueous lumen of the channel denature more rapidly and with higher cooperativity than the surface loop, while sites on the hydrophobic side of the beta-strand undergo a limited degree of noncooperative unfolding and do not fully denature even at high (e.g., 4 M) Gdn-HCl concentrations. We conclude that, in a transmembrane beta-strand, the local environment of a given residue plays a significant role in the loss of structure at each site.

Author List

Klug CS, Feix JB

Authors

Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of Wisconsin
Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Bacterial Outer Membrane Proteins
Carrier Proteins
Cell Membrane
Electron Spin Resonance Spectroscopy
Escherichia coli
Guanidine
Mutagenesis, Site-Directed
Protein Denaturation
Protein Folding
Receptors, Cell Surface
Spin Labels
Thermodynamics