NMR study of the cold, heat, and pressure unfolding of ribonuclease A.
NMR study of the cold, heat, and pressure unfolding of ribonuclease A.
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DOI：
10.1021/bi00027a012
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发表时间：
1995-07
期刊：
影响因子：
2.9
通讯作者：
Jing Zhang;Xiangdong Peng;A. Jonas;J. Jonas
中科院分区：
文献类型：
--
作者：
Jing Zhang;Xiangdong Peng;A. Jonas;J. Jonas
本文用一维和二维核磁共振氢谱研究了核糖核酸酶A及其抑制剂复合物的可逆冷、热、压去折叠。在pH 2.0和10 ℃下，在1巴至5千巴的压力范围内进行可逆压力变性实验。冷变性在3 kbar下进行，其中蛋白质溶液可以冷却至-25 ° C而不冷冻。包括热变性实验，所获得的实验数据使我们能够构建RNase A的压力-温度相图。实验结果表明，所有三个变性过程（冷，热和压力）导致非合作展开的可能性。在冷变性和压力变性的RNase A光谱中出现新的组氨酸共振，与热变性状态下不存在这种共振相比，表明压力变性和冷变性状态可能含有部分折叠的结构，其类似于Blum等人报道的温度跳跃实验中发现的早期折叠中间体的结构[Blum，A. D、等人（1978）J. Mol. 118，305]。进行氢交换实验以确认在压力变性状态下部分折叠结构的存在。在压力变性状态下观察到稳定的氢键结构，保护骨架酰胺氢不受溶剂交换的影响。这些实验结果表明，压力变性的RNase A显示出抑制剂3 '-UMP的特性，表明RNase A-抑制剂复合物比没有抑制剂的RNase更稳定。
The reversible cold, heat, and pressure unfolding of RNase A and RNase A--inhibitor complex were studied by 1D and 2D 1H NMR spectroscopy. The reversible pressure denaturation experiments in the pressure range from 1 bar to 5 kbar were carried out at pH 2.0 and 10 degrees C. The cold denaturation was carried out at 3 kbar, where the protein solution can be cooled down to -25 degrees C without freezing. Including heat denaturation experiments, the experimental data obtained allowed us to construct the pressure--temperature phase diagram of RNase A. The experimental results suggest the possibility that all three denaturation processes (cold, heat, and pressure) lead to non-cooperative unfolding. The appearance of a new histidine resonance in the cold-denatured and pressure-denatured RNase A spectra, compared to the absence of this resonance in the heat-denatured state, indicates that the pressure-denatured and cold-denatured states may contain partially folded structures that are similar to that of the early folding intermediate found in the temperature-jump experiment reported by Blum et al. [Blum, A. D., et al. (1978) J. Mol. Biol. 118, 305]. A hydrogen-exchange experiment was performed to confirm the presence of partially folded structures in the pressure-denatured state. Stable hydrogen-bonded structures protecting the backbone amide hydrogens from solvent exchange were observed in the pressure-denatured state. These experimental results suggest that the pressure-denatured RNase A displays the characteristics of a the inhibitor 3'-UMP show that the RNase A-inhibitor complex is more stable than RNase without the inhibitor.