FOURIER-TRANSFORM INFRARED SPECTROSCOPIC STUDIES OF CA2+-BINDING PROTEINS

The secondary structures of calmodulin and parvalbumin are well established from X-ray diffraction and nuclear magnetic resonance spectroscopic studies, which indicate that these proteins are predominantly alpha-helical in character. Recent infrared studies have nevertheless suggested that the helical structures present in these proteins in solution are not the standard alpha-helix but rather some kind of distorted helices [Trewhella, J., et al. (1989) Biochemistry 28, 1294]. The evidence for this was the unusually low amide I frequency for calmodulin and troponin C in (H2O)-H-2 solution. The studies presented here, however, suggest that the helical structures in these proteins are not significantly distorted, for two reasons. First, distorted helical structures have weaker hydrogen bonds than the standard alpha-helix and would therefore be expected to absorb at a higher rather than a lower frequency. Second, distorted helical structures would absorb at an unusual frequency in H2O solutions which is not the case for the proteins studied here. The band frequency of these proteins is observed to occur at a frequency observed with other proteins known to contain predominantly alpha-helical structures. Quantitative analysis of the FT-IR spectra of calmodulin (67% alpha-helix) and parvalbumin (68% alpha-helix) in H2O in the presence of Ca2+ gives helical contents similar to those reported by X-ray studies. This raises the question as to why these proteins in H2O show a normal frequency for the presence of alpha-helical structures and an abnormal frequency in (H2O)-H-2. Addition of deuterated glycerol to the proteins in (H2O)-H-2 solutions results in a significant shift of absorbance to higher frequency. This is consistent with dehydration of the protein taking place. Circular dichroism spectra of the proteins in glycerol show no evidence of any structural rearrangements. The unusually low amide frequency for these proteins in (H2O)-H-2 is interpreted on the basis of an unusual degree of solvent interaction with exposed helical structures, lowering the amide I maximum only in (H2O)-H-2 due to the increased strength of deuterium bonds as compared to hydrogen bonds. Quantitative analyses of FT-IR spectra of calmodulin and parvalbumin show an increase in the helical content by approximately 6% for both proteins on addition of Ca2+. A reduced rate of hydrogen-deuterium exchange in the Ca2+-loaded state suggests the formation of a more compact structure. The most marked effect of Ca2+ is an enhanced thermal stability of these proteins. Elevation of the temperature to 70-degrees-C in the absence of Ca2+ results in disruption of helical structures and formation of an intermolecular beta-sheet. This rearrangement is prevented by Ca2+.