W-band orientation selective DEER measurements on a Gd 3+ /nitroxide mixed-labeled protein dimer with a dual mode cavity

Date

2013

Authors

Kaminker, Ilia
Tkach, Igor
Manukovsky, Nurit
Yagi, Hiromasa
Bennati, Marina
Goldfarb, Daniella
Otting, Gottfried
Huber, Thomas

Journal Title

Journal ISSN

Volume Title

Publisher

Academic Press

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 μs 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 spin labels.

Description

Keywords

Keywords: DEER; Double electron-electron resonance; Dual modes; Frequency separation; Frequency spacing; High field; Homodimers; Intense signals; Nitroxides; Single mode cavity; Spin label; Structural information; Distance measurement; Paramagnetic resonance; Prote DEER; Distance measurements; Dual mode cavity; EPR; Gd3+ spin labels; High field; Orientation selection

Citation

Source

Journal of Magnetic Resonance

Type

Journal article

Book Title

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DOI

10.1016/j.jmr.2012.11.028

Restricted until

2037-12-31