Kalbitzer H R, Görler A, Li H, Dubovskii P V, Hengstenberg W, Kowolik C, Yamada H, Akasaka K
University of Regensburg, Institute of Biophysics and Physical Biochemistry, Germany.
Protein Sci. 2000 Apr;9(4):693-703. doi: 10.1110/ps.9.4.693.
The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa(-1) at 278 K and 2.20 ppm GPa(-1), respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa(-1). Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the beta-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded beta-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.
利用二维（2D）异核核磁共振光谱，在从大气压到200兆帕的压力范围内，研究了来自肉葡萄球菌的15N富集的组氨酸磷酸载体蛋白（HPr）的压力诱导变化。核磁共振实验能够同时观测主链和侧链酰胺质子及氮原子。随着压力增加，大多数共振峰向低场移动，表明普遍存在压力诱导的构象变化。在278K时，酰胺质子和氮原子的平均压力诱导位移分别为0.285 ppm GPa⁻¹和2.20 ppm GPa⁻¹。在298K时，相应的值分别为0.275和2.41 ppm GPa⁻¹。如果对所有酰胺共振峰进行测定，质子和氮的压力系数显示出显著但相当小的相关性（0.31）。当将分析限制在β折叠片中的酰胺基团时，这些系数之间的相关性明显更高，为0.59。正如已针对其他蛋白质所描述的那样，酰胺质子压力系数与相应氢键距离密切相关，因此是氢键长度压力诱导变化的指标。氮位移变化似乎也能感知其他物理现象，如局部主链构象的变化。根据HPr蛋白的结构变化对压力诱导位移进行解释，得出以下情况：HPr蛋白的四链β折叠片是结构中最不易压缩的部分，仅显示出较小的压力效应。两条长螺旋a和c显示出中间效应，这可以通过更高的可压缩性和螺旋的伴随弯曲来解释。最大的压力系数出现在His15周围的活性中心区域以及包含HPr激酶磷酸化位点Ser46的调节螺旋b中。这表明结构的这一部分存在多种不同的结构状态，其平衡群体因压力而发生变化。与显示出大压力效应的活性中心环周围残基不同，Ile14的质子和氮压力系数非常小。它可能代表活性中心环的某种锚定点，可以将其固定在空间中的正确位置，而环的其他部分则适应不断变化的外部条件。
