Study of the thermal denaturation of ribonuclease A by differential thermal analysis and susceptibility to proteolysis

1. The thermally induced change in conformation of ribonuclease A in solution was investigated by differential thermal analysis and the susceptibility of the enzyme to proteolytic digestion by ficin. 2. A transition with a mid-point of 60.5°C at pH4.2 was observed directly by differential thermal analysis and shown to be a property of the native structure. 3. At pH4.2 ribonuclease A is susceptible to ficin digestion at 60°C but not at 18°C. 4. Chromatographic analysis of the digestion products reveals that transient active intermediates are produced during the digestion. 5. Three of these intermediates were purified and partially characterized. 6. The nature of those sections of the ribonuclease molecule that are involved in the thermal transition is discussed.     

The denaturation of ribonuclease A by combinations of urea and salt denaturants.

When urea is added to ribonuclease A that has already been denatured by salt (CaCl₂, LiClO₄ or LiCl were used), a second co-operative transition occurs, supporting the previous demonstration that these salts cause only partial denaturation. Also we have studied the effect of the salts on the urea denaturation, and the effect of urea on the salt denaturation. At low concentrations urea makes the salt transitions occur at lower concentrations, but at higher concentrations it changes the transition so that the completely disordered protein found in urea is produced by the salt. At low concentrations the salts actually stabilize the protein against denaturation by urea, but at higher concentrations they destabilize it. The data are presented in “phase diagrams” which are found to be very useful for such three-component systems.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Heat denaturation of ribonuclease

One of the main and, chronologically, perhaps one of the first questions in the study of globular protein heat denaturation is that of the applicability of the “all or none” principle to this process, i.e., whether the transition of globular protein from the native into the denaturated state occurs abruptly, without intermediate, thermodynamically stable forms or there are several successive transitions. Despite an intensive study of the process of denaturation this question still remains unsettled. Moreover, its actuality has greatly increased lately with the accumulation of contradictory data.     

Pretransitional structural changes in the thermal denaturation of ribonuclease S and S protein

Two mechanisms have been proposed for the thermal unfolding of ribonuclease S (RNase S). The first is a sequential partial unfolding of the S peptide/S protein complex followed by dissociation, whereas the second is a concerted denaturation/dissociation. The thermal denaturation of ribonuclease S and its fragment, the S protein, were followed with circular dichroism and infrared spectra. These spectra were analyzed by the principal component method of factor analysis. The use of multiple spectral techniques and of factor analysis monitored different aspects of the denaturation simultaneously. The unfolding pathway was compared with that of the parent enzyme ribonuclease A (RNase A), and a model was devised to assess the importance of the dissociation in the unfolding. The unfolding patterns obtained from the melting curves of each protein imply the existence of multiple intermediate states and/or processes. Our data provide evidence that the pretransition in the unfolding of ribonuclease S is due to partial unfolding of the S protein/S peptide complex and that the dissociation occurs at higher temperature. Our observations are consistent with a sequential denaturation mechanism in which at least one partial unfolding step comes before the main conformational transition, which is instead a concerted, final unfolding/dissociation step.     

The denaturation transition of DNA in mixed solvents

The helix-to-coil denaturation transition in DNA has been investigated in mixed solvents at high concentration using ultraviolet light absorption spectroscopy and small-angle neutron scattering. Two solvents have been used: water and ethylene glycol. The "melting" transition temperature was found to be 94 degrees C for 4% mass fraction DNA/d-water and 38 degrees C for 4% mass fraction DNA/d-ethylene glycol. The DNA melting transition temperature was found to vary linearly with the solvent fraction in the mixed solvents case. Deuterated solvents (d-water and d-ethylene glycol) were used to enhance the small-angle neutron scattering signal and 0.1M NaCl (or 0.0058 g/g mass fraction) salt concentration was added to screen charge interactions in all cases. DNA structural information was obtained by small-angle neutron scattering, including a correlation length characteristic of the inter-distance between the hydrogen-containing (desoxyribose sugar-amine base) groups. This correlation length was found to increase from 8.5 to 12.3 A across the melting transition. Ethylene glycol and water mixed solvents were found to mix randomly in the solvation region in the helix phase, but nonideal solvent mixing was found in the melted coil phase. In the coil phase, solvent mixtures are more effective solvating agents than either of the individual solvents. Once melted, DNA coils behave like swollen water-soluble synthetic polymer chains.     

The thermal unfolding of ribonuclease A. A 13C NMR study.

The 13C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1-7 and between 6 and 70 degrees C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as 'two-state', evidence is presented for intermediate unfolding stages at least 10 degrees C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17-24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T1 and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70 degrees C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others. The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.     

X-ray crystallographic studies of the denaturation of ribonuclease S.

In an attempt to view the onset of urea denaturation in ribonuclease we have collected X-ray diffraction data on ribonuclease S crystals soaked in 0, 1.5, 2, 3, and 5 molar urea. At concentrations above 2 M urea, crystals were stabilized by glutaraldehyde crosslinking. We have also collected data on ribonuclease S crystals at low pH in an attempt to study the onset of pH denaturation. The resolution of the datasets range from 1.9 to 3.0 A. Analysis of the structures reveals an increase in disorder with increasing urea concentration. In the 5 M urea structure, this increase in disorder is apparent all over the structure but is larger in loop and helical regions than in the beta strands. The low pH structure shows a very similar pattern of increased disorder. In addition there is a major change in the position of the main chain (> 1 A) in the 65-72 turn region. This region has previously been shown to be involved in one of the initial steps of unfolding in the reduction of ribonuclease A. Crystallographic analyses in the presence of denaturant, when combined with controlled crosslinking, can thus provide detailed structural information that is related to the initial steps of unfolding in solution. Proteins 1999;36:282-294.     

Hydration and thermal denaturation of beta-lactoglobulin. A calorimetric study.

The thermal properties of the beta-lactoglobulin-water system were investigated by differential scanning calorimetry in the temperature range from -50 to 130 degrees C. Determination of the heat and temperature of fusion of the absorbed water allowed resolution of the water into four different states. The amounts of water in these states were different for samples before and after heat denaturation. In the case of denatured beta-lactoglobulin, a smaller amount of water with thermal properties different from ordinary water was observed and its total water binding capacity was lower. The thermal stability of beta-lactoglobulin in the water content range from 0 to 0.75 g/g showed a strong dependence on the degree of hydration. A correlation was observed between the changes in the thermal stability of the protein and the changes in the state of the absorbed water. The results are compared with those obtained from similar measurements of other globular proteins and of fibrillar proteins.     

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.     

Two-phase unfolding pathway of ribonuclease A during denaturation induced by dithiothreitol

The dynamics of the unfolding process of bovine pancreatic ribonuclease A (RNase A) unfolded by dithiothreitol (DTT) at a low concentration of 1:30 were investigated in alkaline phosphate-buffered saline solutions at 303K and 313K by using proton nuclear magnetic resonance ((1)H NMR) spectra. Three NMR spectral parameters including Shannon entropy, mutual information, and correlation coefficient were introduced into the analysis. The results show that the unfolding process of RNase A was slowed to the order of many hours, and the kinetics of the unfolding pathway described by the three parameters is best fit by a model of two consecutive first-order reactions. Temperature greatly influences the rate constants of the unfolding kinetics with different temperature effects observed for the fast and the slow processes. The consistencies and the differences between the three sets of parameters show that they reflect the same relative denaturation pathway but different spectra windows of the unfolding process of RNase A. The results suggest that the unfolding process of RNase A induced by low concentrations of DTT is a two-phase pathway containing fast and slow first-order reactions.     

Characterization of the unfolding of ribonuclease A in aqueous methanol solvents

The effect of methanol on the thermal denaturation of ribonuclease A has been investigated over the -40 to 70 degrees C range. The transition was fully reversible to at least 60% (v/v) methanol at an apparent pH of cryosolvent (pH) of 3.0 and was examined at methanol concentrations as high as 80%. The unfolding transition, as monitored by absorbance change at 286 nm, became progressively broader and occurred at increasingly lower temperatures as the alcohol concentration increased. In 50% methanol, increasing the pH from 2 to 6 shifted the transition to higher temperature. A substantial decrease in cooperativity was noted at the more acidic conditions. On the other hand, increasing concentrations of guanidine hydrochloride in 50% methanol caused the transition to shift to lower temperatures with little effect on the cooperativity. The observed effects on the cooperativity of the unfolding transition suggest that methanol and lower temperatures may increase the concentration of partially folded intermediate states in the unfolding of ribonuclease. Comparison of the transition in 50% methanol as determined by absorbance or fluorescence, which monitor the degree of exposure of buried tyrosines and hence the tertiary structure, to that determined by far-UV circular dichroism, which monitors secondary structure, indicated that the major unfolding transition occurred at a higher temperature in the latter case. Thus, the tertiary structure is lost at a lower temperature than the secondary structure. This observation is consistent with a model of protein folding in which initially formed regions of secondary structure pack together, predominantly by hydrophobic interactions, to give the tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thermal unfolding of ribonuclease A A 13C NMR study

The ¹³C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1–7 and between 6 and 70°C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as ‘two-state’, evidence is presented for intermediate unfolding stages at least 10°C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17–24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T₁ and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70°C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others.The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.     

The heat denaturation of bacterial ribonuclease studied by infrared spectroscopy]

The thermal denaturation of bacterial ribonuclease in the interval of pH 2.5-7.0 has been investigated by means of infra-red spectroscopy method. The protein melting for pH 2.5 begins at the temperature 25 degrees C and is accompanied by secondary protein structure reconstruction, partially destroying native beta-structure and leading to new denatured conformation appearance of different types of beta-turns. Spectral changes for pH 3.5 and 7.0 are significantly less in the same frequency areas. At the temperature more than 50 degrees C protein aggregation takes place with inter-molecule-beta-form formation.     

High resolution thermal denaturation of mammalian DNAs.

High resolution melting profiles of different mammalian DNAs are presented. Melting curves of various mammalian DNAs were compared with respect to the degree of asymmetry, first moment, transition breath and Tmi of individual subtransitions. Quantitative comparison of the shape of all melting curves was made. Correlation between phylogenetical relations among mammals and shape of the melting profiles of their DNAs was demonstrated. The difference between multi-component heterogeneity of mammalian DNAs found by optical melting analysis and sedimentation in CsCl-netropsin density gradient is also discussed.     

Saltatory thermal denaturation of double-stranded viral RNAs.

The double-stranded RNAs from bacteriophage phi6 and the replicative form of mengovirus denature upon heating in a series of abrupt steps which resemble the subtransitions (thermalites) observed within the high resolution profiles of small, naturally occurring DNA molecules. Such RNA thermalites are approximately an order of magnitude narrower than typical thermal subtransitions of nominally single-stranded RNA. We conclude that the same features of nucleotide sequence that give rise to cooperative denaturation in DNA genomes are to be found also in RNA genomes. Thus, high resolution thermal denaturation profiles are useful for characterizing double-stranded RNA molecules as well as native DNA in the size range of common viruses. A medium containing dimethylsulfoxide was required to lower the Tm of the RNA samples to a satisfactory temperature range. For double-stranded RNA in 50% dimethylsulfoxide, the dependence of Tm on G . C composition was greater than that of DNA in the same medium and also greater than that of double-stranded RNA in an aqueous medium. The fact that RNA thermalites are broader than DNA thermalites and that the melting temperature of double-stranded RNA has a greater dependence on base composition than that of DNA, indicates that at least one of the thermodynamic parameters for double helix formation in RNA is different from that in DNA.