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

MPS-Authors
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Tkach,  I.
Research Group of Electron Paramagnetic Resonance, MPI for biophysical chemistry, Max Planck Society;

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Bennati,  M.
Research Group of Electron Paramagnetic Resonance, MPI for biophysical chemistry, Max Planck Society;

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1629151.pdf
(Publisher version), 479KB

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Citation

Kaminker, I., Tkach, I., Manukovsky, N., Huber, T., Yagi, H., Otting, G., et al. (2013). W-band orientation selective DEER measurements on a Gd3+/nitroxide mixed-labeled protein dimer with a dual mode cavity. Journal of Magnetic Resonance, 227, 66-71. doi:10/1016/j.jmr.2012.11.028.


Cite as: http://hdl.handle.net/11858/00-001M-0000-000E-8026-E
Abstract
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 ls in a protein. This opens the possibility of accessing distances of 8 nmand 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 spin labels.