Infrared spectra of amide groups in alpha-helical proteins: Evidence for hydrogen bonding between helices and water

Infrared spectral frequencies of amide vibrational modes are sensitive to s econdary structure. In this work, evidence is presented that accessibility to water additionally affects spectral positions. The dimeric alpha -helica l coiled-coil GCN4-P1' was C-13 labeled in the amide carbonyl groups of bur ied Leu or exposed Ala. At 20 degreesC, the amide I' peak for C-13 Ala amid e is at 1585 cm(-1), whereas the position for C-13 Leu is at 1606 cm(-1). T hese shifts permit the distinction of solvent-exposed and buried amide grou ps. Lowering temperature increases H-bond strength, producing a shift to lo wer frequency. In the temperature range from 10 to 273 K in aqueous glycero l, 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 solven t. In addition, spectral shifts of the amide bands for N-methylacetamide an d the solvent-exposed component of the proteins follow the glass transition temperature of the cryosolvent. In contrast, the amide transitions assigne d to alpha -helical segments that are expected to have little interaction w ith water undergo the weakest shifts. The amide I' band of the alpha -helic al protein parvalbumin also shows subpeaks that shift differently with temp erature, 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 contrib utions from volume expansion of the sample, and variations in the average h ydrogen bond angle, induced by population of low-frequency librational mode s involving the solvent and protein. The results on the isotopically labele d peptides conclusively show that alpha -helical regions that are or are no t solvent exposed can be distinguished both by the position of the amide I' peak and by the temperature-dependent shifts.