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. Study holds ProTherm entries: 4901, 4902 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:27 p.m.
|Number of data points||6|
|Proteins||Ferrienterobactin receptor ; Ferrienterobactin receptor|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence QEPTDTPVSHDDTIVVTAAEQNLQAPGVSTITADEIRKNPVARDVSKIIRTMPGVNLTGNSTSGQRGNNRQIDIRGMGPENTLILIDGKPVSSRNSVRQGWRGERDTRGDTSWVPPEMIERIEVLRGPAAARYGNGAAGGVVNIITKKGSGEWHGSWDAYFNAPEHKEEGATKRTNFSLTGPLGDEFSFRLYGNLDKTQADAWDINQGHQSARAGTYATTLPAGREGVINKDINGVVRWDFAPLQSLELEAGYSRQGNLYAGDTQNTNSDSYTRSKYGDETNRLYRQNYALTWNGGWDNGVTTSNWVQYEHTRNSRIPEGLAGGTEGKFNEKATQDFVDIDLDDVMLHSEVNLPIDFLVNQTLTLGTEWNQQRMKDLSSNTQALTGTNTGGAIDGVSTTDRSPYSKAEIFSLFAENNMELTDSTIVTPGLRFDHHSIVGNNWSPALNISQGLGDDFTLKMGIARAYKAPSLYQTNPNYILYSKGQGCYASAGGCYLQGNDDLKAETSINKEIGLEFKRDGWLAGVTWFRNDYRNKIEAGYVAVGQNAVGTDLYQWDNVPKAVVEGLEGSLNVPVSETVMWTNNITYMLKSENKTTGDRLSIIPEYTLNSTLSWQAREDLSMQTTFTWYGKQQPKKYNYKGQPAVGPETKEISPYSIVGLSATWDVTKNVSLTGGVDNLFDKRLWRAGNAQTTGDLAGANYIAGAGAYTYNEPGRTWYMSVNTHF|