UVRR SPECTROSCOPY OF THE PEPTIDE-BOND .1. AMIDE-S, A NONHELICAL STRUCTURE MARKER, IS A C-ALPHA-H BENDING MODE

Raman spectra with deep UV excitation are reported for N-methylacetamide. In addition to the well-known amide I, II, and III bands, an enhanced band at 1385 cm-1, labeled amide S, is identified as a C-H bending mode of the acetyl group via its disappearance upon C-CD3 substitution. At the same time, the nearby amide III shifts up from 1316 to 1347 cm-1, showing it to be coupled with amide S and thereby accounting for the enhancement of the latter. This assignment is, in addition, supported by the UV resonance Raman spectra of a series of N-methylacetamide derivatives that have different numbers of C-alpha-H's. The ca. 1395-cm-1 UVRR band of peptides is likewise shown to be a C-alpha-H bending mode via C-alpha deuteration of the oligopeptides triglycine and N-acetyltrialanine methyl ester. This band disappears when polylysine is converted to the alpha-helical form at high pH, and likewise when polyglutamate is converted to the alpha-helical form at low pH. Its disappearance is proposed to result from uncoupling of the amide S and amide III modes when the C-alpha-H bond is rotated into an orientation which is cis to the nearby peptide carbonyl group in alpha-helices, from the trans orientation found in beta-sheet or loop structures. The disappearance of amide S upon NH/D exchange in D2O is likewise attributable to uncoupling, due to the large shift in amide III. These amide uncoupling effects as well as the uncoupling of amide II from amide III and amide S in cis-amides are mainly due to kinematic changes in the various conformers, as revealed by normal mode analysis of NMA. Quantitative analysis of UVRR spectra for a range of proteins shows the amide S intensity to be linearly related to the nonhelical content and to extrapolate to zero for 100% alpha-helix, consistent with its behavior in polylysine and polyglutamate. Intensity values are given for amide S and for amide II, which can be used to determine helix content in polypeptides and proteins.