Heterogeneity in the Nitroxide Micro-Environment: Polarity and Proticity Effects in Spin-Labeled Proteins Studied by Multi-Frequency EPR

This study aims to investigate the gxx heterogeneity of the g tensor commonly observed in high field EPR spectra of nitroxide spin labeled sites in proteins. This heterogeneity is addressed in terms of spin label populations characterized by different polarity and H bonding properties of the nitroxide micro environment. The gxx value for each population is determined from the fit of continuous wave high field spectra obtained at 95, 275 and 360 GHz with a series of nitroxide spin labels covalently attached to different sites in both membrane and water soluble proteins. The spin labeled proteins investigated include sensory rhodopsin II SRII and its cognate transducer molecule HtrII from N. pharaonis both in micelles and membranes, bacteriorhodopsin from H. salinarum in native purple membrane lipid bilayers and water soluble colicin A from E. coli. To avoid contributions to the gxx spectral features of the nitroxide label due to nuclear quadrupole interactions arising from 14N nuclei, and to simplify the nitrogen hyperfine pattern, methanethiosulfonate spin labels were employed containing the 15N isotope I 1 2 in some experiments. A consistent analysis of all multi frequency EPR spectra revealed three distinct gxx values, gxx, for each investigated position of the labeled proteins. In contrast, distinctly different nitrogen hyperfine splittings Azz of the nitroxides in the various labeled proteins could not be resolved, but rather an average hyperfine splitting amp; 256;zz was obtained. The gxx values as well as the fractions of the different nitroxide populations were found to be correlated with the average hyperfine constant amp; 256;zz, a parameter which likewise is known to be sensitive to the local polarity of the spin label micro environment. Plotting the different gxx values obtained for each EPR spectrum vs amp; 256;zz of the labeled proteins reveals new interesting aspects of the nitroxide label micro environment in terms of polarity and H bonding propensity proticity . Linear approximations of the different regions of the plot gxx vs amp; 256;zz are presented and compared with theoretical and experimental data available from the literatur