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

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.