AN NMR INVESTIGATION OF THE CONFORMATIONAL EFFECT OF NITROXIDE SPIN LABELS ON ALA-RICH HELICAL PEPTIDES

Nitroxide spin labels, in conjunction with electron spin resonance (ES R) experiments, are extensively employed to probe the structure and dy namics of biomolecules. One of the most ubiquitous spin labeling reage nts is the methanethiosulfonate spin label which attaches a spin label selectively to Cys residues via a disulfide bond (Cys-SL). However, t he actual effect of the nitroxide spin label upon the conformation of the peptide or protein cannot be unambiguously determined by ESR. In t his study, a series of 16-residue Ala-rich helical peptides was charac terized by nuclear magnetic resonance techniques. The C alpha H chemic al shift analysis, NOEs, and (3)J(NH alpha) coupling constants for pep tides with no Cys, free Cys, and Cys-SL (with the N-O group reduced) w ere compared. These results indicate that while replacement of an Ala with a Cys residue causes a loss of overall helical structure, the Cys -SL residue is helix supporting, as would be expected for a non-beta-b ranched aliphatic amino acid. Thus, the Cys-SL residue does not pertur b helical structure and, instead, exhibits helix-stabilizing character istics similar to that found for Ala, Met, and Leu. (C) 1999 Academic Press.