Electron paramagnetic resonance (EPR) site-directed spin labeling (SDSL) is emerging as a valuable tool in structural biology. It has numerous capabilities which have proven useful in probing local areas of large proteins that are inaccessible to other methods. SDSL, which involves the attachment of a sulfhydryl-specific nitroxide spin label to a single free cysteine residue, allows the local area of the labeled site to be studied in detail. We have used a variety of the SDSL techniques to investigate the structure and dynamics of FepA, an 81kDa ligand-gated outer membrane receptor for ferric enterobactin (FeEnt). This protein is proposed to form a beta-barrel structure composed of antiparallel beta-strands and several extracellular loops, the largest of which contains the ligand binding site. Using the continuous wave (CW) power saturation technique and calculating the depths of lipid-exposed residues, we have completed the first mapping of an entire transmembrane beta-strand (Klug et al. (1997) Biochemistry 36, 13027-13033). This proved the beta-barrel proposal for FepA correct. Other studies have focused on the conformational change that occurs in the protein upon FeEnt binding to the extracellular ligand binding domain. This change has been followed by observing spin label motion and accessibility at various residues within both the binding region and along a beta-strand. Electron spin echo (ESE) has been used to examine interaction between a specific spin label and the bound ligand (Klug et al., (submitted) Biochemistry). Finally, we have carried out chemical denaturation work to study the unfolding of FepA at specific sites in the ligand binding loop region and along the rigid beta-barrel (Klug et al., (submitted) Protein Chemistry). The guanidine-HCl-induced unfolding of the loop region and sites in the aqueous channel are highly cooperative while the unfolding of the lipid-exposed beta-strand residues studied do not follow cooperative unfolding. This work indicates that interaction between the beta-barrel residues facing the channel lumen with residues on the ligand-binding surface loop are important to the structural make-up of the receptor and possibly also in transmembrane signaling. Taken together, these studies have provided several new insights into the functional dynamics and structure of FepA that are uniquely accessible to EPR SDSL. ?

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
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Medical College of Wisconsin
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Mao, Li; Liu, Yu-Xiang; Huang, Chun-Hua et al. (2015) Intrinsic Chemiluminescence Generation during Advanced Oxidation of Persistent Halogenated Aromatic Carcinogens. Environ Sci Technol 49:7940-7
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