Heterogeneity in the nitroxide micro-environment: Polarity and proticity effects in spin-labeled proteins studied by multi-frequency EPR

E. Bordignon, H. Brutlach, Alex L Urban, K. Hideg, A. Savitsky, A. Schnegg, P. Gast, Mark H Engelhard, E. J J Groenen, K. Möbius, Heinz Juergen Steinhoff

Research output: Contribution to journalArticleResearchpeer-review

40 Citations (Scopus)


This study aims to investigate the gxx heterogeneity of the g-tensor commonly observed in high-field electron paramagnetic resonance (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 and its cognate transducer molecule (HtrII) from Natronomonas pharaonis both in micelles and membranes, bacteriorhodopsin from Halobacterium salinarum in native purple membrane lipid bilayers and water-soluble colicin A from Escherichia 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, containing the 15N isotope (I = 1/2) in some experiments, were employed. A consistent analysis of all multi-frequency EPR spectra revealed three distinct gxx values, gxx i, 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 Āzz was obtained. The gxx i values as well as the fractions of the different nitroxide populations were found to be correlated with the average hyperfine constant Āzz a parameter which likewise is known to be sensitive to the local polarity of the spin-label micro-environment. Plotting the different gxx i values obtained for each EPR spectrum versus Āzz 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 i versus Āzz are presented and compared with theoretical and experimental data available from the literature.

Original languageEnglish
Pages (from-to)391-403
Number of pages13
JournalApplied Magnetic Resonance
Issue number1
Publication statusPublished - 2010
Externally publishedYes

Cite this