Polarity dependence of EPR parameters for TOAC and MTSSL spin labels: Correlation with DOXYL spin labels for membrane studies

TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) is a nitroxyl amino acid that can be incorporated in the backbone of peptides. DOXYL (4,4-dimethyl-oxazolidine-1-oxyl) is a nitroxyl ring that can be attached rigidly at specific C-atom positions in the acyl chains of phospholipids. Spin-labelled phosphatidylcholines of the DOXYL type have been used previously to establish the transmembrane polarity profile in biological lipid bilayers [D. Marsh, Polarity and permeation profiles in lipid membranes, Proc. Natl. Acad. Sci. USA 87 (2001) 7777–7782]. Here, we determine the polarity dependence of the isotropic 14N-hyperfine couplings, a(0)N, and g-values, g(o), in a wide range of protic and aprotic media, for a TOAC-containing dipeptide (Fmoc-TOAC-Aib-OMe) and for a DOXYL-containing fatty acid (12-DOXYL-stearic acid). The correlation between datasets for TOAC and DOXYL nitroxides in the various solvents is used to establish the polarity profile for isotropic hyperfine couplings of TOAC in a transmembrane peptide. This calibration can be used to determine the location of TOAC at selected residue positions in a transmembrane or surface-active peptide. A similar calibration procedure is also applied to a(0)N and g(o) for the pyrroline methanethiosulphonate nitroxide (MTSSL) that is used in site-directed spin-labelling studies of membrane proteins.