Abstract
Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.
Highlights
IntroductionDistance measurement by double electronelectron resonance (DEER) (double electron-electron resonance, called PELDOR) is an established technique to track macromolecular structures[1]
Distance measurement by double electronelectron resonance (DEER) is an established technique to track macromolecular structures[1]
We assessed the multidrug transporter (Mdr) activity of single cysteine mutants (Fig. S1) and those that retained function were selected for further analyses, including www.nature.com/scientificreports expression tests (Fig. S2A) and MTSSL accessibility (Fig. S2B)
Summary
Distance measurement by DEER (double electron-electron resonance, called PELDOR) is an established technique to track macromolecular structures[1]. In this work we applied DEER with MTSSL, measured at Q-band, and with a Gd(III) spin label, Gd-C2 (Fig. 1) measured at W-band Using these labels we characterized the conformational behavior of the membrane protein MdfA, an Escherichia coli secondary multidrug transporter (Mdr)[60]. This structure shows that, MdfA shares general structural characteristics of major facilitator superfamily (MFS) transporters[65]. Understanding the functional dynamics of MdfA in its natural environment requires accurate distance constraints between regions in the protein that undergo movement during substrate binding and release
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