The spatial effect of protein deuteration on nitroxide spin-label relaxation: Implications for EPR distance measurement

Hassane El Mkami,Richard Ward,Andrew Bowman,Tom Owen-Hughes,David G Norman

doi:10.1016/j.jmr.2014.09.010

Abstract

Pulsed electron–electron double resonance (PELDOR) coupled with site-directed spin labeling is a powerful technique for the elucidation of protein or nucleic acid, macromolecular structure and interactions. The intrinsic high sensitivity of electron paramagnetic resonance enables measurement on small quantities of bio-macromolecules, however short relaxation times impose a limit on the sensitivity and size of distances that can be measured using this technique. The persistence of the electron spin-echo, in the PELDOR experiment, is one of the most crucial limitations to distance measurement. At a temperature of around 50K one of the predominant factors affecting persistence of an echo, and as such, the sensitivity and measurable distance between spin labels, is the electron spin echo dephasing time (Tm). It has become normal practice to use deuterated solvents to extend Tm and recently it has been demonstrated that deuteration of the underlying protein significantly extends Tm. Here we examine the spatial effect of segmental deuteration of the underlying protein, and also explore the concentration and temperature dependence of highly deuterated systems.

Highlights

In a non-deuterated environment, short spin echo dephasing times (Tm) [1,2,3], in the order of 2–4 ls, are usually observed, when studying nitroxide spin-labeled proteins, in frozen solution at around 50 K
The estimated Tm for the nondeuterated, octameric complex is 6.9 ls, which is at the high end of reported Tm values for a spin label situated on the surface of a protein dissolved in deuterated buffer [1]
Deuteration of H3 leads to an approximate doubling of the Tm to

Summary

Introduction

In a non-deuterated environment, short spin echo dephasing times (Tm) [1,2,3], in the order of 2–4 ls, are usually observed, when studying nitroxide spin-labeled proteins, in frozen solution at around 50 K. Tm is affected by contributions from instantaneous and spectral diffusion as well as hyperfine interactions with surrounding nuclei. Unpaired electrons can show dipolar coupling to nuclear spins in the surrounding media and individual nuclear spin flip is slow, the large number of coupled nuclei in a typical protein makes these events highly probable and spin flips in dipolar coupled nuclei change the precession frequency of the unpaired electron. Dipolar coupling is proportional to the magnetic moment, so proton spin diffusion is a more effective mechanism of

Present address

Sample preparation

Calculation of spatial relationships between spin label and protons

Results

Discussion and conclusion