ESR-SPECTRA REFLECT LOCAL AND GLOBAL MOBILITY IN A SHORT SPIN-LABELED PEPTIDE THROUGHOUT THE ALPHA-HELIX-]COIL TRANSITION

A series of short alanine-based synthetic peptides (16 or 17 residues) have previously been shown to exhibit an anomalously high degree of alpha-helicity [Marqusee, S., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5286-5290; Marqusee, S., & Baldwin, R. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8898-8902]. These peptides are ideal models for extracting position-dependent structural and dynamic information. Using the methanethiosulfonate nitroxide spin label (MTSSL), we labeled an analogue of the salt-bridge-stabilized "i+4" peptide, called the "i+4c", which has a specific attachment site created by replacing the central alanine with a cysteine. Circular dichroism (CD) spectra demonstrate that the i+4c-MTSSL peptide retains nearly the same helicity as the original i+4 peptide. The ESR spectra of the labeled peptide indicate no significant aggregation. ESR spectra were acquired throughout the helix-coil transition by temperature variation. From the motionally narrowed spectra, we extracted the rotational correlation times of the nitroxide label. Parallel measurements with circular dichroism enabled us to relate these parameters directly to the fractional helicity. For comparison, we followed a similar procedure with MTSSL-labeled glutathione (GS-MTSSL), a tripeptide that does not form an alpha-helix. Our results are interpreted in terms of a local tumbling volume, V(L), which reflects the portion of the peptide that reorients with the nitroxide label. At high fractional helicity, V(L) is similar to the volume expected for a 17-residue helix. Only when the fractional helicity is lowered below 40% does V(L) decrease, thereby indicating the onset of segmental motion of the peptide backbone. We analyze these results in terms of both a cooperative and a noncooperative model of the alpha-helix --> coil transition, and we propose that the simplest cooperative view is not complete. Our experimental results and analysis show that spin label ESR is an excellent tool for rapidly extracting important structural information from nanomole quantities of peptide.