Maleimide, Iodoacetamide, Indanedione, and Chloromercuric Spin Label Reagents with Derivatized Nitroxide Rings as ESR Reporter Groups for Protein Conformation and Dynamics

Abstract

The syntheses of eight nitroxide spin labels which bear maleimide, iodoacetamide, indanedione, or chloromercuric reactive groups and, in addition, a second substituent in the nitroxide ring are presented. The second substituent groups range from hydrophobic and hydrophilic esters to carboxylic acid and secondary and tertiary amine groups. The resulting spin labels are characterized with respect both to protein covalent modification and to the electron spin resonance spectral properties of the bound labels. The effect of the various substituents in the spin label on the reactivity toward the membrane-bound shark rectal gland and pig kidney Na,K-ATPase is described. The spectral differences between immobilized and mobile groups observed by electron spin resonance for the different protein-bound spin labels show that, by selecting an appropriate derivative for modification, a large range of different motional sensitivities of the reporter group can be obtained. Such different series of spin labels should therefore be useful for detecting mobility changes arising from conformational transitions in proteins by conventional electron spin resonance spectroscopy or for measurement of protein rotational diffusion using saturation transfer electron spin resonance spectroscopy. The chloromercuric series is found to be particularly useful because of the high reactivity, the lack of reversibility that potentially is associated with the Michael addition reaction, and the wide range of rotational mobility that is exhibited by the different derivatives.