DEER and RIDME Measurements of the Nitroxide-Spin Labelled Copper-Bound Amine Oxidase Homodimer from Arthrobacter Globiformis

Abstract

In the study of biological structures, pulse dipolar spectroscopy (PDS) is used to elucidate spin–spin distances at nanometre-scale by measuring dipole–dipole interactions between paramagnetic centres. The PDS methods of Double Electron Electron Resonance (DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME) are employed, and their results compared, for the measurement of the dipolar coupling between nitroxide spin labels and copper-II (Cu(II)) paramagnetic centres within the copper amine oxidase from Arthrobacter globiformis (AGAO). The distance distribution results obtained indicate that two distinct distances can be measured, with the longer of these at c.a. 5 nm. Conditions for optimising the RIDME experiment such that it may outperform DEER for these long distances are discussed. Modelling methods are used to show that the distances obtained after data analysis are consistent with the structure of AGAO.

In situ distance measurements in a membrane transporter using maleimide functionalized orthogonal spin labels and 5-pulse electron double resonance spectroscopy

“…Spectroscopically orthogonal labels can provide more information per sample as compared to an identical spin pair. Following a first demonstration of this possibility using 14 N/ 15 N labeling, several examples including the triple labeling using Gd(III), Mn(II), and NO tags have been reported [75][76][77][78][79][80][81][82][83]. In the Gd(III)-NO case, faster relaxation and a higher transition moment for Gd(III) enable the spectroscopic separation.…”

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms

“…This can be overcome by relaxation-induced dipolar modulation enhancement (RIDME). RIDME (Figure B) relies on the coupled center undergoing longitudinal relaxation to modulate the signal of the coupled spin centers. , The background contribution in a RIDME experiment is higher, which, therefore, affects the modulated echo signal. At the same time, artifacts arising from improper phase cycles can also affect the acquired results.…”

DEER Data Analysis Software: A Comparative Guide

“…The data were all from nitroxide Q-band 4-pulse DEER and the samples are the copper amine oxidase protein from Arthrobacter globiformis (AGAO) and spin-labelled DNA. Experimental details are given in the relevant publications and the data are freely available with links presented in the publications ( Hardwick et al, 2020 ; Russell et al, 2021 ).…”