Infrared Spectra of Amide Groups in α-Helical Proteins:  Evidence for Hydrogen Bonding between Helices and Water

Infrared spectral frequencies of amide vibrational modes are sensitive to secondary structure. In this work, evidence is presented that accessibility to water additionally affects spectral positions. The dimeric α-helical coiled-coil GCN4-P1‘ was 13C labeled in the amide carbonyl groups of buried Leu or exposed Ala. At 20 °C, the amide I‘ peak for 13C Ala amide is at 1585 cm-1, whereas the position for 13C Leu is at 1606 cm-1. These shifts permit the distinction of solvent-exposed and buried amide groups. Lowering temperature increases H-bond strength, producing a shift to lower frequency. In the temperature range from 10 to 273 K in aqueous glycerol, the amide transitions assigned to solvent-exposed regions of the helices undergo the strongest temperature-dependent shifts, similar to that of the peptide bond model compound, N-methylacetamide, in the same aqueous solvent. In addition, spectral shifts of the amide bands for N-methylacetamide and the solvent-exposed component of the proteins follow the glass transition temperature of the cryosolvent. In contrast, the amide transitions assigned to α-helical segments that are expected to have little interaction with water undergo the weakest shifts. The amide I‘ band of the α-helical protein parvalbumin also shows subpeaks that shift differently with temperature, and on the basis of their temperature dependence and frequency can be assigned to solvent exposed or buried regions. The spectral shifts are discussed in terms of changes in hydrogen bond strengths, including contributions from volume expansion of the sample, and variations in the average hydrogen bond angle, induced by population of low-frequency librational modes involving the solvent and protein. The results on the isotopically labeled peptides conclusively show that α-helical regions that are or are not solvent exposed can be distinguished both by the position of the amide I‘ peak and by the temperature-dependent shifts.