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

Hannah Russell,Janet E Lovett,Rachel Stewart,Christopher Prior,Thembaninkosi G Gaule,Vasily S Oganesyan

doi:10.1007/s00723-021-01321-6

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.

Highlights

In the study of biological structures, pulsed dipolar spectroscopy (PDS) can be a valuable tool
This work has demonstrated the applicability of the PDS methods Double Electron Electron Resonance (DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME) in the measurement between nitroxide R1 spin centres and bound paramagnetic Cu(II) ions in the AGAO homodimeric protein
Both PDS methods achieved results which were in excellent agreement with each other, and with an AGAO model produced by molecular dynamics (MD) simulation and the rotamer library approach

Summary

Introduction

In the study of biological structures, pulsed dipolar spectroscopy (PDS) can be a valuable tool. Sophisticated and computationally demanding molecular dynamics (MD) simulations allow for construction of a protein model from its primary sequence and the addition of the spin label for explicit inclusion in calculations (with the optional inclusion of experimentally derived structural restraints). Such MD simulations enable the time evolution of complex biomolecular systems with introduced spin labels and probes to be monitored and analysed in relation to their EPR spectra [9,10,11]

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