TEMPERATURE COEFFICIENTS OF AMIDE PROTON NMR RESONANCE FREQUENCIES INTRIFLUOROETHANOL - A MONITOR OF INTRAMOLECULAR HYDROGEN-BONDS IN HELICAL PEPTIDES

2D H-1 NMR spectroscopy of two a-helical peptides which differ in thei r amphipathicity has been used to investigate the relationships betwee n amide-proton chemical shifts, amide-proton exchange rates, temperatu re, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and t emperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from -10 to -6 ppb/K, usually indic ative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the alpha-helices. Howev er, slow hydrogen isotope exchange for residues from 4 to 16 in both 1 8-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observation s, we suggest that the pattern of amide-proton shifts in a-helices in H2O/TFE solvents is dominated by bifurcated intermolecular hydrogen-bo nd formation between the backbone carbonyl groups and TFE. The amide-p roton chemical shift changes with increasing temperature may be interp reted by a disruption of intermolecular hydrogen bonds between carbony l groups and the TFE in TFE/water rather than by the length of intramo lecular hydrogen bonds in alpha-helices.