Reinstädler D, Fabian H, Backmann J, Naumann D
Robert Koch-Institut, Berlin, Germany.
Biochemistry. 1996 Dec 10;35(49):15822-30. doi: 10.1021/bi961810j.
We undertook a first detailed comparative analysis of the refolding kinetics of ribonuclease A (RNase A) by time-resolved Fourier transform infrared spectroscopy. The refolding process was initiated either by applying a temperature jump on the thermally denatured protein or by rapid dilution of a concentrated [13C]urea solution containing the chemically unfolded protein. The dead time of the injecting and mixing devices and the time-resolution of the spectrometer permitted us to monitor the refolding kinetics in a time range of 100 ms to minutes. The infrared amide I' band at 1631 cm-1 was used to directly probe the formation of beta-sheet structure during the refolding process. The aromatic ring stretching vibration of tyrosine at 1515 cm-1 was employed as a local monitor that detects changes in the tertiary structure along the folding pathway. The comparative analysis of the kinetics of the beta-sheet formation of chemically and thermally denatured ribonuclease A revealed similar folding rates and amplitudes when followed under identical refolding conditions. Therefore, our kinetic infrared studies provide evidence for a high structural similarity of urea-denatured and heat-denatured RNase A, corroborating the conclusions derived from the direct comparison of the infrared spectra of thermally and chemically denatured RNase A under equilibrium conditions [Fabian, H., & Mantsch, H.H. (1995) Biochemistry 34, 13651-13655]. In detail, the kinetic infrared data demonstrate that in the time window of 0.1-30 s approximately 40% of the native beta-sheet structure in RNase A is formed in the presence of 0.6 M urea at pH* 3.6, indicating that up to 60% of the beta-structure is formed out of the time window used in this study. Temperature jump experiments in the absence of chemical denaturants exhibited faster and more complex refolding kinetics. In addition, differences in the time constants of refolding derived from the amide I' band at 1631 cm-1 and from the tyrosine vibration at 1515 cm-1 were observed, indicating that the formation of secondary structure precedes the formation of stable tertiary contacts during the refolding of RNase A.
我们通过时间分辨傅里叶变换红外光谱对核糖核酸酶A（RNase A）的重折叠动力学进行了首次详细的比较分析。重折叠过程可通过对热变性蛋白进行温度跃升，或通过快速稀释含有化学变性蛋白的浓缩[13C]尿素溶液来启动。注射和混合装置的死时间以及光谱仪的时间分辨率使我们能够在100毫秒至数分钟的时间范围内监测重折叠动力学。1631厘米-1处的红外酰胺I'带用于直接探测重折叠过程中β-折叠结构的形成。1515厘米-1处酪氨酸的芳香环伸缩振动被用作局部监测器，以检测沿折叠途径三级结构的变化。对化学变性和热变性核糖核酸酶A的β-折叠形成动力学的比较分析表明，在相同的重折叠条件下进行跟踪时，其折叠速率和幅度相似。因此，我们的动力学红外研究为尿素变性和热变性RNase A的高度结构相似性提供了证据，证实了在平衡条件下对热变性和化学变性RNase A的红外光谱进行直接比较得出的结论[法比安，H.，&曼茨，H.H.（1995年）《生物化学》34卷，13651 - 13655页]。详细地说，动力学红外数据表明，在0.1 - 30秒的时间窗口内，在pH* 3.6、0.6 M尿素存在的情况下，RNase A中约40%的天然β-折叠结构形成，这表明高达60%的β-结构是在本研究使用的时间窗口之外形成的。在没有化学变性剂的情况下进行的温度跃升实验显示出更快且更复杂的重折叠动力学。此外，观察到从1631厘米-1处的酰胺I'带和1515厘米-1处的酪氨酸振动得出的重折叠时间常数存在差异，这表明在RNase A的重折叠过程中，二级结构的形成先于稳定三级接触的形成。
