We have investigated the effect of deuteration of non‐exchangeable protons on protein global thermal stability, hydrophobicity, and local flexibility using well‐known thermostable model systems such as the villin headpiece subdomain (HP36) and the third immunoglobulin G‐binding domain of protein G (GB3). Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) measurements as a function of temperature probe global thermal stability in the presence of acetonitrile, while differential scanning calorimetry determines thermal stability in solution. Both indicate small but measurable changes in the order of several degrees. RP‐HPLC also permitted quantification of the effect of deuteration of just three core phenylalanine side chains of HP36. NMR dynamics investigation has focused on methyl axes motions using cross‐correlated relaxation measurements. The analysis of order parameters provided a complex picture indicating that deuteration generally increases motional amplitudes of sub‐nanosecond motion in GB3 but decreases those in HP36. Combined with earlier dynamics measurements at C_α–C_β sites and backbone sites of GB3, which probed slower time scales, the results point to the need to probe multiple atoms in the protein and variety of time scales to the discern the full complexity of the effects of deuteration on dynamics.

中文翻译：

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蛋白质上不可交换质子的氘化影响其热稳定性，侧链动力学和疏水性。

我们使用著名的热稳定模型系统（例如villin Headpiece子域（HP36）和蛋白质G的第三个免疫球蛋白G结合域）研究了不可交换质子氘化对蛋白质全局热稳定性，疏水性和局部柔韧性的影响（GB3）。反相高效液相色谱（RP-HPLC）测量与温度探针在乙腈存在下的整体热稳定性有关，而差示扫描量热法则确定溶液的热稳定性。两者都表示微小但可测量的变化，变化幅度为几度。RP-HPLC还允许仅对HP36的三个核心苯丙氨酸侧链的氘化效果进行定量。NMR动力学研究集中在使用互相关弛豫测量的甲基轴运动上。顺序参数的分析提供了一张复杂的图片，表明氘化通常会增加GB3中亚纳秒运动的运动幅度，但会降低HP36中的运动幅度。结合早期的C动态测量_α -C _β位点和GB3的骨干位点，其探测慢的时间尺度，结果指出，需要探测的蛋白质和各种时间尺度的多原子与辨别的氘化的动力学的影响的全部复杂。