An NMR Investigation of the Conformational Effect of Nitroxide Spin Labels on Ala-Rich Helical Peptides

✍ Scribed by Kimberly A. Bolin; Paul Hanson; Sarah J. Wright; Glenn L. Millhauser

• Publisher: Elsevier Science
• Year: 1998
• Tongue: English
• Weight: 151 KB
• Volume: 131
• Category: Article
• ISSN: 1090-7807
• DOI: 10.1006/jmre.1998.1365

✦ Synopsis

Nitroxide spin labels, in conjunction with electron spin reso- (1)(2)(3). In addition to probing the nature of the equilibrium nance (ESR) experiments, are extensively employed to probe the between aand 3 10 -helices in linear peptides (4-6), MTSSL structure and dynamics of biomolecules. One of the most ubiquihas been used to probe a variety of systems, ranging from tous spin labeling reagents is the methanethiosulfonate spin label linear peptides (7) to integral membrane proteins (8,9). Shin which attaches a spin label selectively to Cys residues via a disuland co-workers recently developed a ''spectroscopic ruler'' fide bond (Cys-SL). However, the actual effect of the nitroxide for determining distances from 8 to 25 A ˚using the deconvoluspin label upon the conformation of the peptide or protein cannot tion of dipolar-coupled spectra of model Ala-rich a-helical be unambiguously determined by ESR. In this study, a series of peptides ( 10). This method should be applicable to numerous 16-residue Ala-rich helical peptides was characterized by nuclear systems, including large or membrane-bound proteins. Other magnetic resonance techniques. The C a H chemical shift analysis, work on peptide systems by Cafiso and co-workers used ESR NOEs, and 3 J NHa coupling constants for peptides with no Cys, to determine the orientation and insertion depth of alamethicin, free Cys, and Cys-SL (with the N-O group reduced) were coma voltage-gated channel forming peptide, with respect to the pared. These results indicate that while replacement of an Ala

with a Cys residue causes a loss of overall helical structure, the membrane surface (7, 11). ESR results from a series of pep-Cys-SL residue is helix supporting, as would be expected for a tides labeled with MTSSL at various positions parallel recent non-b-branched aliphatic amino acid. Thus, the Cys-SL residue solid-state NMR results and confirm that alamethicin inserts does not perturb helical structure and, instead, exhibits helix-stabiwith the helix axis normal to the lipid bilayer, the N-terminus lizing characteristics similar to that found for Ala, Met, and Leu. completely buried within the membrane, and the C-terminus ᭧ 1998 Academic Press in the aqueous region approximately 4 A ˚from the membranesolution interface (7). Some of the most extensive work utilizing MTSSL is being carried out by Hubbell and co-workers