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

Abstract

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 (14)N-hyperfine couplings, a(o)(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(o)(N) and g(o) for the pyrroline methanethiosulphonate nitroxide (MTSSL) that is used in site-directed spin-labelling studies of membrane proteins.

Molecular Dynamics and Partitioning of Di‐tert‐butyl Nitroxide in Stratum Corneum Membranes: Effect of Terpenes

“…The a 0 parameters were measured at 25°C for the spin labels DTBN and DOXYL (5-DOXYL stearic acid) in terpenes and other solvents, with varying dielectric constants (data not shown) and the a 0 values found for DTBN were on average 1.0 higher than DOXYL. Similar results were reported recently in a study comparing the a 0 values of several spin labels with those of DOXYL class [30]. Thus, with the subtraction of 1.0 G from the a 0 values presented in Fig.…”

Reaction fields and solvent dependence of the EPR parameters of nitroxides: The microenvironment of spin labels

“…Fig. 5 shows the dependence of a N o on relative dielectric permittivity of the solvent for the DTBN [3] and 12-SASL spin labels [29]. All solvents considered are aprotic, i.e., are not functioning as hydrogen-bond donors (cf.…”

“…The polarisability of the nitroxide is included in every case, and is found to have a particularly significant effect for the Wertheim model. An analysis such as this is important not only for comparison of spin-labelled biological environments with solutions of defined polarity, but also for transfer of polarity dependences between different nitroxide spin labels [28,29]. The latter is particularly significant for transmembrane polarity profiles that are established mostly by oxazolidine-nitroxide spin-labelled lipids [30][31][32], whereas integral membrane peptides and proteins are mostly spin-labelled with piperidine-, pyrrolidine-or pyrroline-nitroxide derivatives (e.g., Refs.…”

Nitroxide Radicals: Properties, Synthesis and Applications

“…47 determined the polarity dependence of the isotropic 14 N-hyperfine couplings, a N , and g-values in a wide range of protic and aprotic media, for a TOAC-containing dipeptide 31 and for a DOXYL-containing fatty acid 32 (Scheme 5.11). The correlation between data sets for TOAC and DOXYL nitroxides in various solvents was used to establish the polarity profile for isotropic hyperfine couplings of TOAC in a transmembrane peptide.…”