DENATURANT UNFOLDING OF THE FERRIC ENTEROBACTIN RECEPTOR AND LIGAND-INDUCED STABILIZATION STUDIED BY SITE-DIRECTED SPIN-LABELING

FepA is an integral outer membrane protein that is the specific recept or for the siderophore, ferric enterobactin, and is thus primarily res ponsible for iron uptake in many Gram-negative bacteria. A site-specif ic mutant of FepA, containing a single introduced cysteine in the liga nd-binding domain, was spin labeled and used to examine the denaturant -induced unfolding of this receptor with guanidine hydrochloride (Gdn- HCl) and urea. Electron spin resonance (ESR) spectra showed conversion of the spin label from a motionally-restricted, immobilized environme nt to a freely-accessible, rotationally-mobile state upon denaturation . Unfolding was also followed by nondenaturing polyacrylamide gel elec trophoresis (PAGE), which is sensitive to loss of the putative transme mbrane beta-structure, and displayed a similar concentration dependenc e. Unfolding occurred over relatively narrow ranges of denaturant conc entration, indicating a high degree of cooperativity. Unfolding was fu lly reversible under the conditions employed. Rapid, spontaneous refol ding occurred in the presence of Triton X-100 and did not require exog enous lipids. Refolding could be induced by either dialysis, dilution to low denaturant concentration, or ethanol precipitation. At ambient temperature the free energy of unfolding extrapolated to zero denatura nt concentration (Delta G(u)(o)) was 6.24 +/- 0.63 kcal/mol. Values of Delta G(u)(o) obtained with Gdn-HCl and urea were in good agreement, as were values obtained from linear extrapolation and nonlinear regres sion fitting to a two-state equilibrium, This is the first report of a quantitative evaluation of the free energy of unfolding for an integr al membrane protein.