Thermal Denaturation Of Ribonuclease Research Articles

Thermal denaturation of ribonuclease A (RNase A) at different concentrations of Guanidinium hydrochloride (GuHCl) and urea

Thermal denaturation of ribonuclease A (RNase A) at different concentrations of Guanidinium hydrochloride (GuHCl) and urea

Clarification of the thermally-induced pretransition of ribonuclease A in solution by principal component analysis and two-dimensional correlation infrared spectroscopy

Thermal denaturation of ribonuclease A (RNase A) in D 2O solution is studied by Fourier transform infrared (FT-IR) spectroscopy. Sample–sample two-dimensional correlation (SS 2D) spectroscopy and principal component analysis (PCA) are applied to these spectral data to reveal the thermal kinetics of RNase A. The second scores plot of PCA constructed from temperature-dependent original IR spectra illustrates a pretransition at 46 °C as well as a clear main transition at 66 °C. The latter is revealed by the SS 2D correlation spectra and the first score of PCA because of their illustration of the main denaturation event of RNase A, while the former cannot. Therefore, the present study demonstrates the great potential of PCA in revealing subtle phase transition of proteins in aqueous solutions.

4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization

The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM< c<250 mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9.

Calorimetric evidence for conformational transitions of RNase A in the presence of cytidine 2′,3′-cyclic phosphate

Thermal denaturation of ribonuclease A (RNase A) complex with cytidine 3′-monophosphate (3′CMP) was studied by differential scanning calorimetry (DSC). The kinetic and binding studies of RNase A with cytidine 2′,3′-cyclic phosphate (cCMP) as a substrate, and 3′CMP as a ligand were also investigated by difference spectrophotometry. The obtained kinetic saturation curve reveals the occurrence of an anomalous non-hyperbolic shape at high substrate concentrations, and a biphasic binding isotherm. These phenomena indicate that a conformational change is occurring with RNase A during the hydrolysis of cCMP. A combination of kinetic and thermodynamic studies tends to elucidate the reasons for the formation of a non-hyperbolic behavior in a kinetic saturation curve. The thermal profile of the enzyme-3′CMP complexes shows a splitting of two distinct peaks with different structural stabilities of melting points ( T m) of 325 and 337 K. The bifurcate appearance of DSC profile of RNase A-3′CMP complexes manifests a physical view of a light kinetic structural transition. It is worthy to note, the direct binding (not via enzymatic reaction) of enzyme with 3′CMP indicates single DSC profile and monophasic binding isotherm.

Calorimetric evidence for conformational transitions of RNase A in the presence of cytidine 2′,3′-cyclic phosphate

Thermal denaturation of ribonuclease A (RNase A) complex with cytidine 3′-monophosphate (3′CMP) was studied by differential scanning calorimetry (DSC). The kinetic and binding studies of RNase A with cytidine 2′,3′-cyclic phosphate (cCMP) as a substrate, and 3′CMP as a ligand were also investigated by difference spectrophotometry. The obtained kinetic saturation curve reveals the occurrence of an anomalous non-hyperbolic shape at high substrate concentrations, and a biphasic binding isotherm. These phenomena indicate that a conformational change is occurring with RNase A during the hydrolysis of cCMP. A combination of kinetic and thermodynamic studies tends to elucidate the reasons for the formation of a non-hyperbolic behavior in a kinetic saturation curve. The thermal profile of the enzyme-3′CMP complexes shows a splitting of two distinct peaks with different structural stabilities of melting points (Tm) of 325 and 337 K. The bifurcate appearance of DSC profile of RNase A-3′CMP complexes manifests a physical view of a light kinetic structural transition. It is worthy to note, the direct binding (not via enzymatic reaction) of enzyme with 3′CMP indicates single DSC profile and monophasic binding isotherm.

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.

Thermodynamics of thermal denaturation of ribonuclease A and cytochrome c in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol

The thermal denaturation of ribonuclease A and cytochrome c has been studied by differential scanning calorimetry (d.s.c.) and u.v.-visible spectrophotometry in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at pH = 5.5 and pH = 4.0, respectively. The quantitative thermodynamic parameters accompanying the thermal transitions from native to denatured state have been evaluated. The results of the reversible thermal denaturations have been fitted with a two-state native-to-denatured mechanism. A comparison has been made of the relative effect of HFIP on the thermal stability of ribonuclease A and cytochrome c. It has been observed that the denaturation capacity of HFIP tends more towards cytochrome c compared with ribonuclease A. The results have been explained on the basis of a fine balance between the preferential exclusion and binding that take place during the course of the denaturation reaction and the structuring of water around the groups of the protein exposed upon denaturation. Using the thermodynamic data obtained from calorimetric and spectroscopic measurements, we have calculated the changes in preferential solvation of ribonuclease A and cytochrome c upon heat denaturation. It is observed that the preferential solvation of these two proteins is specific, indicating that the solvation mechanism is not the same for them.

Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model.

The thermal denaturation of ribonuclease A (RNase A) in the presence of phosphate at neutral pH was studied by differential scanning calorimetry (DSC) and a combination of optical spectroscopic techniques to probe the existence of intermediate states. Fourier transform infrared (FTIR) spectra of the amide I' band and far-uv circular dichroism (CD) spectra were used to monitor changes in the secondary structure. Changes in the tertiary structure were monitored by near-uv CD. Spectral bandshape changes with change in temperature were analyzed using factor analysis. The global unfolding curves obtained from DSC confirmed that structural changes occur in the molecule before the main thermal denaturation transition. The analysis of the far-uv CD and FTIR spectra showed that these lower temperature-induced modifications occur in the secondary structure. No pretransition changes in the tertiary structure (near-uv CD) were observed. The initial changes observed in far-uv CD were attributed to the fraying of the helical segments, which would explain the loss of spectral intensity with almost no modification of spectral bandshape. Separate analyses of different regions of the FTIR amide I' band indicate that, in addition to alpha-helix, part of the pretransitional change also occurs in the beta-strands.

Thermal Denaturation of Ribonuclease A Characterized by Water 17O and 2H Magnetic Relaxation Dispersion

Water oxygen-17 and deuteron nuclear magnetic relaxation dispersion (NMRD) measurements were used to characterize ribonuclease A (RNase A) in the course of thermal denaturation at pH 2 and 4. The structure and dynamics of the protein were probed by specific long-lived water molecules, by the short-lived surface hydration, and by labile side-chain hydrogens. The NMRD data show that native RNase A contains at least three water molecules with a mean residence time of 8 ns at 27 degreesC and an activation enthalpy of ca. 40 kJ mol-1. These water molecules are identified with some or all of six ordered water molecules partly buried in surface pockets in the crystal structure of RNase A. The loss of the 17O dispersion at higher temperatures demonstrates that, in the thermally denatured protein, these surface pockets are either not present or undergoing large structural fluctuations on a subnanosecond time scale. The relaxation dispersion step vanishes monotonically and essentially in concert with the CD denaturation curves, thus ruling out the existence of equilibrium intermediates with a substantial amount of non-native and long-lived hydration water. The NMRD data show that thermally denatured RNase A has a relatively compact but highly flexible structure. The global solvent exposure and the hydrodynamic volume of the denatured protein are much less than for maximally unfolded disulfide-intact RNase A. The NMRD data show that thermal denaturation is accompanied by a large reduction of the mean-square orientational order parameter of side-chain O-H bonds, implying that, in the denatured state, these side chains sample a wide distribution of conformational states on a subnanosecond time scale.

An osmolyte effect on the heat capacity change for protein folding.

We have carried out a differential scanning calorimetry study into the pH effect on the thermal denaturation of ribonuclease A at several concentrations of the osmolyte sarcosine. In order to properly analyze these data, we have elaborated the thermodynamic theory of the linkage between temperature, cosolvent, and pH effects. The denaturation heat capacity increases with sarcosine concentration. The effects of temperature and sarcosine concentration on the denaturation enthalpy and entropy values are well described by convergence equations, with convergence temperatures of around 100 degrees C for the enthalpy and around 112 degrees C for the entropy; we suggest that these effects might be related to a solvent-induced alteration of the apolar-group-hydration contribution to the folding thermodynamics. From our data, we estimate that about 70 extra molecules of water are thermodynamically bound upon ribonuclease denaturation in diluted aqueous solutions of sarcosine; this number is 6-9 times smaller than that predicted on the basis of the following two premises: (a) the osmolyte is strongly excluded from the surface of both the native and the denatured protein and (b) the denatured state is a fully solvated chain. We suggest that at least one of these two premises does not hold. We briefly comment on the potential use of cosolvent effects on thermal denaturation to evaluate the degree of hydration of denatured proteins (thus providing an independent measure of the consequence of their possible residual structure) and, also, on the possibility of finding substances that are more efficient protein stabilizers than known osmolytes are.

Effect of osmoregulatory solutes on the stability of proteins

The thermal denaturation of ribonuclease A and lysozyme in aqueous solutions of osmoregulatory solutes methylamines [trimethylamine N-oxide, betaine (trimethylammonioacetate) and sarcosine] and urea have been investigated by differential scanning calorimetry (DSC). The transition temperature, heat capacity and enthalpy of denaturation in aqueous solutions of ribonuclease A and lysozyme at pH 6.00 have been determined by a least-squares fit of the excess heat capacity data to the two-state model. Thermodynamic functions of denaturation, ΔG°, ΔH° and ΔS° at various temperatures have also been evaluated. The methylamines increase the thermal stability of ribonuclease A and lysozyme in the order : trimethylamine N-oxide > sarcosine > betaine. The stabilizing effect of methylamines when compared with that of amino acids follows the order : trimethylamine N-oxide > glycine > β-alanine > γ-aminobutyric acid > sarcosine > serine > α-alanine > betaine > proline. The structure-stabilizing effect of methylamines and structure-destabilizing effect of urea on ribonuclease A are nearly additive. No net effect on the stability of ribonuclease A is observed in a 2 : 1 mixture of urea and methylamines.

Enzyme thermoinactivation in anhydrous organic solvents

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.

Thermodynamic nonideality as a probe of reversible protein unfolding effected by variations in pH and temperature: Studies of ribonuclease

Thermodynamic nonideality arising from the space-filling effect of added sucrose is employed to confirm that the reversible unfolding of ribonuclease A effected by acid may be described as an equilibrium between native and unfolded states of the enzyme. However, the extent of the volume change is far too small for the larger isomer to be the fully expanded state, a result signifying that the acid-mediated unfolding of ribonuclease does not conform with the two-state equilibrium model of protein denaturation. Although the thermal denaturation of ribonuclease A is characterized by a larger increase in volume, quantitative reappraisal of published results on the effects of glycerol on this transition at pH 2.8 (Gekko, K., and Timasheff, S. N., 1981 Biochemistry 20, 4677–4686) leads to an estimated volume increase that is much smaller than that inferred from hydrodynamic studies—a disparity attributed to the dual actions of glycerol as a space-filling solute and as a ligand that binds preferentially to the thermally unfolded form of the enzyme. Even in this unfavorable circumstance the fact that glycerol exerts a net excluded volume effect at least confirms that the thermal unfolding of ribonuclease A is an equilibrium transition between two discrete states. The strengths and limitations of using thermodynamic nonideality as a probe of the two-state equilibrium model of protein denaturation are discussed in the light of these findings.

Interaction of cytidine 3'-monophosphate and uridine 3'-monophosphate with ribonuclease a at the denaturation temperature.

Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3'-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding constants of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 +/- 8 M-1 (pH 7.0) to 1410 +/- 54 M-1 (pH 5.0), while the binding enthalpies increase from 17 +/- 13 kJ mol-1 (pH 4.7) to 79 +/- 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 +/- 1 M-1 (pH 7.0) to 402 +/- 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 +/- 5 kJ mol-1 (pH 6.0) to 37 +/- 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 +/- 37 M-1 (pH 6.5) to 2200 +/- 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 +/- 7 kJ mol-1 (pH 6.5). the denaturation transition cooperativities of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Use of high-speed size-exclusion chromatography for the study of protein folding and stability.

The urea denaturation of sperm whale myoglobin and thermal denaturation of ribonuclease have been studied by following the associated volume changes by size-exclusion chromatography on a Toya Soda TSK 3000SW gel permeation column. The permeation properties of the gel have been shown to be invariant in the following solvent systems: 0.2 M NaCl; 8.0 M urea-0.2 M NaCl; and 6.0 M guanidinium chloride ( GdmCl ). A precise measurement of the volume changes associated with solvent-induced protein denaturation is thus practicable. The column was calibrated in the above solvent systems by using 12 well-characterized proteins as standards. In the case of the denaturation of myoglobin by urea, the rate of equilibration of folded and unfolded species is slow on the time scale of the chromatographic experiment, and the two forms are well separated on the column in the transition region. Both the folded and unfolded species are shown to undergo significant swelling in urea. This result suggests that the view of denaturation based solely on the preferential solvation of the unfolded protein is incorrect. The rate of interconversion between folded and unfolded ribonuclease is fast relative to the time scale of the chromatographic experiments performed in this study. This is reflected in the fact that only one peak is observed in the elution profiles of ribonuclease in the transition region. Thermally unfolded ribonuclease has a smaller volume than the unfolded state in urea or GdmCl , suggesting that it has residual structure. The van't Hoff delta H for the thermal unfolding of ribonuclease calculated from the size-exclusion chromatographic experiments (36 +/- 3 kcal/mol) is significantly lower than previously reported values.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetric investigation of ribonuclease thermal denaturation.

Thermal denaturation of ribonuclease has been investigated by scanning microcalorimetric, spectrophotometric and polarimetric techniques. It is shown that the temperature changes of ribonuclease are of a complicated character and may be subdivided by the temperature region into two qualitatively different stages: (a) the predenaturational stage, at which neither abrupt changes in physico‐chemical properties nor heat absorption occur, but where the protein partial heat capacity considerably grows; (b) the denaturational stage which is accompanied by intensive heat absorption with the abrupt change in all physico‐chemical properties. Judging by the coincidence of the calorimetric and effective enthalpies, there is a transition at this stage between the two states without intermediate thermodynamically stable forms. Enthalpy values of denaturation, temperature of denaturation and denaturational changes of the heat capacity at various pH values are given.

Thermal Effects on the Circular Dichroism Spectra of Ribonuclease A and of Ribonuclease S-Protein

The thermal denaturation of ribonuclease A and of S-protein in neutral aqueous solutions has been studied by means of their circular dichroism spectra. For RNase A, the positive circular dichroism extremum near 241 mµ shows a biphasic dependence upon temperature, a linear change between 15° and 50° being followed by a sharp one above 52°. The negative circular dichroism extremum at 277 mµ exhibits only a sharp change at the higher temperatures and appears unchanged between 10° and 50°. Upon cooling of the solution from 65°, enzymic activity is fully recovered, as is the initial magnitude of the 277 mµ extremum, but the ellipticity of the cooled solution at 241 mµ is lower. A positive circular dichroism extremum at 237 mµ is observed in spectra of S-protein below 25°, as well as a negative circular dichroism extremum at 277 mµ; both of these extrema disappear upon heating. In contrast to RNase A, the thermal changes observed for S-protein are not reversible.