BioNMR - W-band orientation selective DEER measurements on a Gd3+/nitroxide mixed-labeled protein dimer with a dual mode cavity

W-band orientation selective DEER measurements on a Gd3+/nitroxide mixed-labeled protein dimer with a dual mode cavity

Available online 12 December 2012
Publication year: 2012
Source:Journal of Magnetic Resonance

Double electron-electron resonance (DEER) at W-band (95 GHz) was applied to measure the distance between a pair of nitroxide and Gd3+ chelate spin labels, about 6 nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700 MHz) between the narrow, intense signal of the Gd3+ central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12 ?s in a protein. This opens the possibility of accessing distances of 8 nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd3+-nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide labels.
Graphical abstract

http://origin-ars.els-cdn.com/conten...003722-fx1.jpg Highlights

? A Gd3+-nitroxide distance of 5.75 nm was determined by W-band DEER. ? A new dual mode cavity gave the required ~700 MHz observe-pump frequency separation. ? 12 ?s dipolar evolution was achieved, distances of 8 nm and above can be accessed. ? Orientation selection was resolved and analyzed.


Source: Journal of Magnetic Resonance