NMR study of the cold, heat, and pressure unfolding of ribonuclease A.
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.
Zhang
J
J
Department of Chemistry, School of Chemical Sciences, University of Illinois, Urbana 61801, USA.
Peng
X
X
Jonas
A
A
Jonas
J
J
D000818:Animals; D002417:Cattle; D003080:Cold Temperature; D006358:Hot Temperature; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D011312:Pressure; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013816:Thermodynamics
D006859:Hydrogen; D012259:Ribonuclease, Pancreatic