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.

This work investigates the relative role of the pure geometrical confinement and the strength of the surface effect on the dynamics of liquids in porous silica glasses prepared by the sol-gel process. The deuteron NMR spin-lattice relaxation times TI of several molecular liquids in porous silica glasses are reported as function of pore size in the range from 18 to 143 A over the temperature range from 260 to 310 K. Molecular liquids studied include strongly interacting polar liquids such as pyridine-d 5 , aniline-d s , and nitrobenzene-d s , whereas the saturated cyclic hydrocarbon liquids of cyclohexane-d 12 and cis-decalin-d ls represent the weakly interacting liquids. In a first approximation, toluene-d l and dioxane-ds are chosen as examples of liquids with intermediate interactions with the silica surface. The experimental relaxation data are analyzed by using the two-state, fast-exchange model which is found to be valid for the strongly interacting liquids and liquids with intermediate interactions. In terms of this model, the viscosity of the surface layer for pyridine-d 5 is about 30 times higher than that for bulk liquid pyridine. The importance of the two-dimensional approach to describe motional dynamics of liquids confined to pores smaller than 30 A is illustrated in the case of weakly interacting liquid of cyclohexane-d I2 . Additional information on the relative role of surface interactions and the pure topological effects on the dynamics of liquids in confined geometries was obtained by using surface-modified glasses in which the surface hydroxyl groups were replaced by OSi(CH 3 )20C2HS groups. Indeed, the effects of surface modifications on the 2H T 1-I are most pronounced for strongly interacting liquids whereas they are absent for cyclohexane. In agreement with the concept of two-dimensional behavior of liquids in small pores, one finds that the low-frequency relaxation times, namely, the spin-spin relaxation time T 2 , and the spin-lattice relaxation time in the rotating coordinate frame, Tip' remain unchanged by surface modification. In fact, this is a consequence of logarithmic enhancement of the spectral density at low frequencies so that the effect of pure geometrical confinement on the T 2-I and T 1-;' I relaxation rates is much larger than any relaxation rate changes arising from surface modification. Several selected NMR TI experiments on pyridine-d s confined to anopore and zeolites are also presented. 6892

Proton and deuteron NMR spin–lattice relaxation times in liquid water and heavy water were measured as a function of pressure and temperature in the range 10–90°C and 1 bar–9 kbar. D2O was also studied at 150 and 200°C. Availability of density and viscosity data under these experimental conditions enabled us to separate the effects of temperature and density on the spin–lattice relaxation times, T1, and viscosities. Under the assumption that the intermolecular dipolar contribution to the proton T1 follows the changes in shear viscosity with temperature and density, we separated the intramolecular and intermolecular dipolar contributions to the proton T1. We found that at a temperature of 10°C the initial increase in density leads to faster reorientation of the water molecules. The effect was much smaller at 30°C. Analysis of the experimental data on H2O and D2O leads to the conclusion that compression diminishes the coupling between the rotational and translational motions of water molecules. The change in the nature of the rotation–translation coupling with increasing density is mainly responsible for the failure of the Debye equation to describe the density effects on the reorientation of water molecules. In the case of D2O we find a relatively small variation in the deuteron quadrupole coupling constant with increasing density. Its average value is approximately 230 kHz over the range of our experimental conditions. Another experimental finding of this study is the decrease in the activation energies for relaxation and shear viscosity with increasing density. All the experimental evidence indicates that compression of water leads to significant distortion and/or disruption of the hydrogen bond network with the important consequence that the dynamic behavior of water under high compression resembles more that of a ’’normal’’ molecular liquid of comparable molecular size. At high densities the hard core repulsive interactions begin to dominate over the directional interactions which are mainly responsible for the open structure of liquid water at low temperatures and pressures.

in 2-1 electrolytes. The model fitting to the experimental data allows us to extract two independent parameters,« and /exptl. The growth behavior of micelles in 2-1 electrolytes is quite different from that in 1-1 electrolytes. The divalent counterions Mg2+ and Ca2+ are more effective in the shielding of electrostatic interactions between micelles than the monovalent counterion Li+. The effective micellar surface charges are strongly affected by the added 2-1 electrolytes when the detergent concentration is low. The measured /exptl is in reasonable agreement with the EMN theory.The mechanism which is responsible for switching the intermicellar interaction from a repulsive to an attractive one is probably due to the gradual dominance of the van der Waals interaction as the double layer repulsion diminishes, and a further investigation on the nature of this attraction should be of great interest.13Acknowledgment. We are grateful to the Biology Department of BNL for use of their small angle spectrometer in this work. Acknowledgement is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this work.

The relative roles of surface and topological effects on the nuclear relaxation rates T−11, T−12, and T−11ρ of polar or nonpolar liquids in porous sol-gel silica glasses are identified via their very different pore size and frequency dependences. On the basis of theory, experimental relaxation rates, and molecular dynamics simulations for the modeled porous systems, the 1/Ti’s are interpreted in terms of a linear combination of bulk, confinement, and surface effects: 1/Ti = 1/Tibulk + ai/R2+ bi/R, where R is the average pore size and ai and bi are given in terms of the usual relaxation parameters of the studied molecular species. This simple expression which allows the determination of the relative roles of surface and topological effects has been used to fit the observed 1H NMR relaxation rates as a function of pore size and frequency for methylcyclohexane, nitrobenzene, pyridine, and toluene both for nonmodified and surface modified porous silica glasses. Using this method, the surface (∝1/R) and pure geometrical (∝1/R2) relaxation contributions are evaluated and the surface and translational correlation times are calculated. More generally, the experimental data allow us to explain the following seemingly paradoxical results obtained for confined liquids: (i) The pure confinement effect is independent of the polarities of the liquid molecules in pores and is very sensitive to the frequency. (ii) The finding of the frequency variation of T−11 and T−11ρ both for polar or nonpolar liquids confined to small pores, shows that the geometrical confinement effects dominate over the surface interaction effects at low frequency and for small pores.