Dzwolak W, Kato M, Shimizu A, Taniguchi Y
Department of Chemistry, College of Science and Engineering, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan.
Biochim Biophys Acta. 1999 Aug 17;1433(1-2):45-55. doi: 10.1016/s0167-4838(99)00150-8.
The Fourier-transform infrared spectroscopy (FTIR) technique with a diamond anvil cell has been applied for examination of the pressure-induced changes occurring in the secondary structure of the alpha-lactalbumin. This is the first high-pressure FTIR study of a calcium-binding protein which simultaneously takes into account spectral changes in both the calcium-ion-binding carboxyl groups' band and the amide I/I' vibrational band. Spectral behavior of three kinds of the protein: the undeuterated holoform, the fully deuterated holoform, and the undeuterated apoform was compared in the pressure range from 0.1 MPa up to 740 MPa. We found that the binding of calcium remarkably stabilizes the alpha-lactalbumin against pressure as it is followed approximately by a 200-MPa increase of the value of pressure at which denaturation occurs. A quantitative analysis of the band of antisymmetrical stretching vibrations of the calcium-binding carboxyl groups revealed that the pressure-induced changes in the calcium-binding loop occur in two stages. Binding of the calcium ion seemingly increases the pressure-stability of the calcium-binding loop to a higher degree than the pressure-stability of the secondary structure of the alpha-lactalbumin. We have also discussed in detail the complex pressure-enhanced H/D exchange in the alpha-lactalbumin. Finally, we have proposed a new assignment of major peaks in the helical region of the amide I/I' spectral band of the partially deuterated alpha-lactalbumin.
采用傅里叶变换红外光谱（FTIR）技术结合金刚石对顶砧池，对α-乳白蛋白二级结构中压力诱导的变化进行了研究。这是首次对钙结合蛋白进行高压FTIR研究，该研究同时考虑了钙离子结合羧基带和酰胺I/I'振动带的光谱变化。在0.1 MPa至740 MPa的压力范围内，比较了三种蛋白质的光谱行为：未氘代的全蛋白形式、完全氘代的全蛋白形式和未氘代的脱辅基蛋白形式。我们发现，钙的结合显著增强了α-乳白蛋白对压力的稳定性，因为变性发生时的压力值大约增加了200 MPa。对钙结合羧基的反对称伸缩振动带进行定量分析表明，钙结合环中压力诱导的变化分两个阶段发生。钙离子的结合似乎比α-乳白蛋白二级结构的压力稳定性更能提高钙结合环的压力稳定性。我们还详细讨论了α-乳白蛋白中复杂的压力增强H/D交换。最后，我们对部分氘代α-乳白蛋白酰胺I/I'光谱带螺旋区域的主要峰提出了新的归属。
