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 receptor for the siderophore, ferric enterobactin, and is thus primarily responsible for iron uptake in many Gram-negative bacteria. A site-specific mutant of FepA, containing a single introduced cysteine in the ligand-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 environment to a freely-accessible, rotationally-mobile state upon denaturation. Unfolding was also followed by nondenaturing polyacrylamide gel electrophoresis (PAGE), which is sensitive to loss of the putative transmembrane beta-structure, and displayed a similar concentration dependence. Unfolding occurred over relatively narrow ranges of denaturant concentration, indicating a high degree of cooperativity. Unfolding was fully reversible under the conditions employed. Rapid, spontaneous refolding occurred in the presence of Triton X-100 and did not require exogenous 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 denaturant concentration (delta GU zero) was 6.24 +/- 0.63 kcal/mol. Values of delta GU zero obtained with Gdn-HCl and urea were in good agreement, as were values obtained from linear extrapolation and nonlinear regression fitting to a two-state equilibrium. This is the first report of a quantitative evaluation of the free energy of unfolding for an integral membrane protein.
Klug
C S
CS
Biophysics Research Institute, Medical College of Wisconsin, Milwaukee 53226, USA.
Su
W
W
Liu
J
J
Klebba
P E
PE
Feix
J B
JB
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004758:Enterobactin / Q000378:metabolism*; D007501:Iron / Q000737:chemistry*; D008024:Ligands; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011956:Receptors, Cell Surface; D013113:Spin Labels; D013816:Thermodynamics
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D008024:Ligands; D011956:Receptors, Cell Surface; D013113:Spin Labels; C029489:enterobactin receptor; D004758:Enterobactin; D007501:Iron