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Biophys J. 2010 Mar 17;98(6):L18-20. doi: 10.1016/j.bpj.2009.12.4193.

Increased sensitivity and extended range of distance measurements in spin-labeled membrane proteins: Q-band double electron-electron resonance and nanoscale bilayers.

Author information

1
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee.

Abstract

We report a significant methodological advance in the application of double electron-electron resonance (DEER) spectroscopy to measure long-range distances in spin-labeled membrane proteins. In the pseudo two-dimensional environment of proteoliposomes, a steep intermolecular background shapes DEER signals leading to long accumulation times, complicating data analysis and reducing the maximal measurable distances from 70 A down to approximately 40-50 A. To eliminate these limitations, we took advantage of the homogeneity and monodispersity of a class of discoidal nanoscale phospholipid bilayers in conjunction with the micromolar DEER sensitivity at Q-band (34 GHz) microwave frequency. Spin-labeled mutants of the ABC transporter MsbA were functionally reconstituted at a ratio of one functional dimer per nanoscale apolipoprotein-bound bilayer (NABB). DEER echo intensities from NABB-reconstituted MsbA have linear baselines reflecting a three-dimensional spatial distribution. This results in an order-of-magnitude higher sensitivity at Q-band relative to proteoliposomes and restores the maximal observable distance effectively increasing experimental throughput. The advances described here set the stage for the use of DEER spectroscopy to analyze conformational dynamics of sample-limited eukaryotic membrane proteins.

PMID:
20303847
PMCID:
PMC2849076
DOI:
10.1016/j.bpj.2009.12.4193
[Indexed for MEDLINE]
Free PMC Article

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