Panick G, Winter R
Department of Chemistry, Physical Chemistry I, University of Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany.
Biochemistry. 2000 Feb 22;39(7):1862-9. doi: 10.1021/bi992176n.
In this paper, we illustrate the use of high-pressure Fourier transform infrared (FT-IR) spectroscopy to study the reversible presssure-induced unfolding and refolding of ribonuclease A (RNase A) and compare it with the results obtained for the temperature-induced transition. FT-IR spectroscopy monitors changes in the secondary structural properties (amide I' band) or tertiary contacts (tyrosine band) of the protein upon pressurization or depressurization. Analysis of the amide I' spectral components reveals that the pressure-induced denaturation process sets in at 5. 5 kbar at 20 degrees C and pH 2.5. It is accompanied by an increase in disordered structures while the content of beta-sheets and alpha-helices drastically decreases. The denatured state above 7 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as an extended random coil. Increase of pH from 2.5 to 5.5 has no influence on the structure of the pressure-denatured state; it slightly changes the stability of the protein only. All experimental evidence indicates that the pressure-denatured states of monomeric proteins have more secondary structure than the temperature-denatured states. Different modes of denaturation, including pressure, may correlate differently with the roughness of the energy scale and slope of the folding funnel. For these reasons we have also carried out pressure-jump kinetic studies of the secondary structural evolution in the unfolding/refolding reaction of RNase A. In agreement with the theoretical model presented by Hummer et al. [(1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1552-1555], the experimental data show that pressure slows down folding and unfolding kinetics (here 1-2 orders of magnitude), corresponding to an increasingly rough landscape. The kinetics remains non-two-state under pressure. Assuming a two-step folding scenario, the calculated relaxation times for unfolding of RNase A at 20 degrees C and pH 2.5 can be estimated to be tau(1) approximately 0.7 min and tau(2) approximately 17 min. The refolding process is considerably faster (tau(1) approximately 0.3 min, tau(2) approximately 4 min). Our data show that the pressure stability and pressure-induced unfolding/refolding kinetics of monomeric proteins, such as wild-type staphylococcal nuclease (WT SNase) and RNase A, may be significantly different. The differences are largely due to the four disulfide bonds in RNase A, which stabilize adjacent structures. They probably lead to the much higher denaturation pressure compared to SNase, and this might also explain why the volume change of WT SNase upon unfolding is about twice as large.
在本文中，我们阐述了使用高压傅里叶变换红外（FT - IR）光谱法研究核糖核酸酶A（RNase A）可逆压力诱导的去折叠和再折叠过程，并将其与温度诱导转变的结果进行比较。FT - IR光谱法可监测蛋白质在加压或减压时二级结构特性（酰胺I'带）或三级结构接触（酪氨酸带）的变化。对酰胺I'光谱成分的分析表明，在20℃和pH 2.5条件下，压力诱导的变性过程在5.5 kbar时开始。此过程伴随着无序结构的增加，而β - 折叠和α - 螺旋的含量则急剧减少。然而，高于7 kbar的变性状态仍保留一定程度的β样二级结构，且分子不能被描述为伸展的无规卷曲。将pH从2.5提高到5.5对压力变性状态的结构没有影响；只是略微改变了蛋白质的稳定性。所有实验证据表明，单体蛋白质的压力变性状态比温度变性状态具有更多的二级结构。不同的变性模式，包括压力变性，可能与能量尺度的粗糙度和折叠漏斗的斜率有不同的相关性。基于这些原因，我们还对RNase A去折叠/再折叠反应中二级结构的演变进行了压力跃变动力学研究。与Hummer等人提出的理论模型[(1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1552 - 1555]一致，实验数据表明压力减缓了折叠和去折叠动力学（此处为1 - 2个数量级），这对应于越来越粗糙的能量景观。在压力下动力学仍非两态。假设为两步折叠过程，在20℃和pH 2.5条件下，RNase A去折叠的计算弛豫时间估计为τ(1)约0.7分钟，τ(2)约17分钟。再折叠过程要快得多（τ(1)约0.3分钟，τ(2)约4分钟）。我们的数据表明，单体蛋白质如野生型葡萄球菌核酸酶（WT SNase）和RNase A的压力稳定性以及压力诱导的去折叠/再折叠动力学可能有显著差异。这些差异主要归因于RNase A中的四个二硫键，它们稳定相邻结构。这可能导致其变性压力比SNase高得多，这也可能解释了为什么WT SNase去折叠时的体积变化大约是其两倍。
