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Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels.
- Source :
-
Scientific reports [Sci Rep] 2019 Aug 29; Vol. 9 (1), pp. 12528. Date of Electronic Publication: 2019 Aug 29. - Publication Year :
- 2019
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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.
- Subjects :
- Cytoplasm chemistry
Cytoplasm genetics
Cytoplasm metabolism
Electron Spin Resonance Spectroscopy
Escherichia coli chemistry
Escherichia coli genetics
Escherichia coli Proteins genetics
Escherichia coli Proteins metabolism
Gadolinium chemistry
Membrane Transport Proteins genetics
Membrane Transport Proteins metabolism
Nitrogen Oxides chemistry
Protein Conformation
Escherichia coli metabolism
Escherichia coli Proteins chemistry
Membrane Transport Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 9
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 31467343
- Full Text :
- https://doi.org/10.1038/s41598-019-48694-0