Guanidine hydrochloride unfolding of a transmembrane beta-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 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.
Klug
C S
CS
Biophysics Research Institute, Medical College of Wisconsin, Milwaukee 53226, USA.
Feix
J B
JB
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D011956:Receptors, Cell Surface; D013113:Spin Labels; D013816:Thermodynamics
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D011956:Receptors, Cell Surface; D013113:Spin Labels; C029489:enterobactin receptor; D019791:Guanidine