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. Study holds ProTherm entries: 9143, 9144, 9145, 9146, 9147, 9148, 9149 Extra Details: ESR; FepA; GdnHCl unfolding; membrane protein stability;,SDSL; transmembrane beta-strand
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:37 p.m.
|Number of data points||21|
|Proteins||Ferrienterobactin receptor ; Ferrienterobactin receptor|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: ddG_H2O|
|Libraries||Mutations for sequence QEPTDTPVSHDDTIVVTAAEQNLQAPGVSTITADEIRKNPVARDVSKIIRTMPGVNLTGNSTSGQRGNNRQIDIRGMGPENTLILIDGKPVSSRNSVRQGWRGERDTRGDTSWVPPEMIERIEVLRGPAAARYGNGAAGGVVNIITKKGSGEWHGSWDAYFNAPEHKEEGATKRTNFSLTGPLGDEFSFRLYGNLDKTQADAWDINQGHQSARAGTYATTLPAGREGVINKDINGVVRWDFAPLQSLELEAGYSRQGNLYAGDTQNTNSDSYTRSKYGDETNRLYRQNYALTWNGGWDNGVTTSNWVQYEHTRNSRIPEGLAGGTEGKFNEKATQDFVDIDLDDVMLHSEVNLPIDFLVNQTLTLGTEWNQQRMKDLSSNTQALTGTNTGGAIDGVSTTDRSPYSKAEIFSLFAENNMELTDSTIVTPGLRFDHHSIVGNNWSPALNISQGLGDDFTLKMGIARAYKAPSLYQTNPNYILYSKGQGCYASAGGCYLQGNDDLKAETSINKEIGLEFKRDGWLAGVTWFRNDYRNKIEAGYVAVGQNAVGTDLYQWDNVPKAVVEGLEGSLNVPVSETVMWTNNITYMLKSENKTTGDRLSIIPEYTLNSTLSWQAREDLSMQTTFTWYGKQQPKKYNYKGQPAVGPETKEISPYSIVGLSATWDVTKNVSLTGGVDNLFDKRLWRAGNAQTTGDLAGANYIAGAGAYTYNEPGRTWYMSVNTHF|