Tamura A, Matsushita M, Naito A, Kojima S, Miura K I, Akasaka K
Graduate School of Science and Technology, Kobe University, Japan.
Protein Sci. 1996 Jan;5(1):127-39. doi: 10.1002/pro.5560050116.
Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme-recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core. The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild-type SSI was deuterium-labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8-1.0 x 10(-10)s indicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 x 10(-8)s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 x 10(-8)s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side-chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein-water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein-water), became extremely high in amplitude and frequency (> 10 MHz). In contrast, the polycrystalline wild-type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side-chain motions are similar in the fully hydrated powder and in crystals. Spin-lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between 10(-8) and 10(-10)s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large-amplitude motions are absent, but the side chains of methionines 70 and/or 73 are likely to have small-amplitude motions.
链霉菌枯草杆菌蛋白酶抑制剂（SSI）的一个亚基中含有三个甲硫氨酸残基：两个分别位于关键的酶识别位点P1和P4（第73位和第70位），另一个（甲硫氨酸103）位于疏水核心区域。通过溶液、晶体和粉末固体状态下的氘核磁共振光谱研究了这三个甲硫氨酸残基侧链的运动以及与枯草杆菌蛋白酶结合时的流动性变化。为此，野生型SSI在所有三个甲硫氨酸残基的甲基上进行了氘标记，并且三个人工突变蛋白分别仅在一个特定的甲硫氨酸甲基上进行了标记。在溶液中，对于甲硫氨酸73和70，有效相关时间仅为0.8 - 1.0×10⁻¹⁰秒，表明表面的两个侧链几乎可以自由波动。然而，与枯草杆菌蛋白酶形成复合物时，这些高流动性被淬灭，甲硫氨酸70和73侧链的相关时间变为1.1×10⁻⁸秒。位于疏水核心区域的甲硫氨酸103在游离SSI中的相关时间至少为1.0×10⁻⁸秒，表明其侧链运动受到高度限制。通过粉末和晶体样品的氘核磁共振光谱更详细地研究了这三个甲硫氨酸侧链的内部运动性质。粉末样品的光谱模式严重依赖于水合作用：冻干后，三个甲硫氨酸残基的侧链运动几乎被淬灭。随着逐渐水合至每克蛋白质 - 水0.20克水，甲硫氨酸70和73甲基轴的取向波动在幅度和频率上都有选择性地增强（至约1兆赫兹），并且在接近饱和水合（每克蛋白质 - 水0.60克水）时，幅度和频率变得极高（> 10兆赫兹）。相比之下，多晶野生型SSI光谱显示出精细结构，反映了甲硫氨酸侧链的特征运动。在最终水合水平下，用于模拟粉末光谱的相同运动模型和参数可以相当好地重现多晶光谱，这表明在完全水合的粉末和晶体中侧链运动相似。自旋晶格弛豫测量表明，即使在晶体中，所有三个甲硫氨酸残基的甲基轴也经历快速运动，相关时间在10⁻⁸至10⁻¹⁰秒之间，与溶液中的相关时间相当。最后，在固态下SSI与枯草杆菌蛋白酶BPN'的水合化学计量复合物中，不存在大幅度运动，但甲硫氨酸70和/或73的侧链可能有小幅度运动。
