Gd3+-Nitroxide Pairs for Protein-Membrane Studies: A Multifrequency EPR Approach

Abstract

The objective of this research project is to advance the well-established method of Site-Directed Spin Labeling (SDSL) Electron Paramagnetic Resonance (EPR) by utilizing many advantages that High Field EPR (HF EPR) could offer. Specifically, we propose to utilize nitroxide-Gd3+ label pairs for site-directed distance measurements in protein and protein-membrane structure studies. The proposed method is based on through space dipole-dipole interactions between Gd3+ ion positioned on the surface of the bilayer and a nitroxide label attached at the point of interest on the lipid molecule or membrane associated protein. The proposed labeling approach coupled with multifreqency EPR can provide information on Gd3+-nitroxide distance, as well as orientation of Gd3+. Experiments were conducted to investigate the mechanism of nitroxide-Gd3+ interactions in solutions of various viscosities at multiple magnetic fields (corresponding frequencies from 9.5 to 220 GHz) in order to determine relative contributions of dipole-dipole and exchange interactions in nitroxide-Gd3+ pairs at various viscosities and to elucidate the relaxation processes modulating the dipole-dipole interaction as a function of magnetic field. Applications for membrane and membrane protein studies are also discussed.