Guanidine hydrochloride unfolding of a transmembrane β-strand in FepA using site-directed spin labeling

Open Access
Publisher Website
Google Scholar
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 β-strand located in the ferric enterobactin receptor, FepA. In addition, we have continued the characterization of the β-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 β-barrel motif comprised of a series of antiparallel β-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 β-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 β-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 β-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 β-strand, the local environment of a given residue plays a significant role in the loss of structure at each site.