NMR-STUDY OF THE COLD, HEAT, AND PRESSURE UNFOLDING OF RIBONUCLEASE-A

被引:214
作者
ZHANG, J
PENG, XD
JONAS, A
JONAS, J
机构
[1] UNIV ILLINOIS,SCH CHEM SCI,DEPT CHEM,URBANA,IL 61801
[2] UNIV ILLINOIS,COLL MED,DEPT BIOCHEM,URBANA,IL 61801
关键词
D O I
10.1021/bi00027a012
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The reversible cold, heat, and pressure unfolding of RNase A and RNase A-inhibitor complex were studied by 1D and 2D H-1 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 molten globule, The cold, heat, and pressure denaturation experiments on the complex of RNase A with the inhibitor 3'-UMP show that the RNase A-inhibitor complex is more stable than RNase without the inhibitor.
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页码:8631 / 8641
页数:11
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