Increased Sensitivity and Range of Distance Measurements in Spin Labeled Membrane Proteins

Abstract

We report a significant methodological advance in the application of double electron-electron resonance (DEER) to spin-labeled membrane proteins. DEER is an unparalleled tool in structural biology yielding long range distance restraints that can be used to model protein folds, to define the nature of conformational changes and determine their amplitudes. Distances are obtained in native-like environments in the absence of conformational selectivity imposed by the crystal lattice and regardless of the molecular mass. However, the realization of these advantages in proteolipsomes has so far lead to significant reduction in the distance range and loss of sensitivity compromising experimental throughput. In the two-dimensional environment of a liposome, the background of intermolecular dipolar spin coupling leads to a strong decay that can obscure the contribution of intramolecular coupling rendering the DEER signals uninterpretable. We found that the combination of two emerging technologies, Q-band pulsed electron paramagnetic resonance and Nanodiscs phospholipid bilayers, overcome the factors limiting DEER sensitivity and distance range. Spin labeled mutants of the ABC transporter MsbA were functionally reconstituted into Nanodiscs at a ratio of one dimer per lipid bilayer. In comparison to proteolisposmes, DEER data from Nanodiscs have a linear baseline reflecting the three dimensional spatial distribution of MsbA. The order of magnitude increase in absolute sensitivity at Q-band microwave frequency is critical given limited sample quantities and working concentrations in the 10-30 μM range. We took advantage of the higher throughput to demonstrate that the magnitude of the distance changes in the ATP hydrolysis cycle is not affected by the lipid headgroup. The advances described here set the stage for the use of DEER spectroscopy to analyze the conformational dynamics of eukaryotic membrane proteins.