Francesca Catanzano

Prediction of the heat capacity change on thermal denaturation of globular proteins

The large positive heat capacity change is a common feature of both the transfer of nonpolar compounds to water and the temperature-induced denaturation of globular proteins. In this paper we present a model for the calculation of the denaturation heat capacity change, that is a key parameter of protein thermodynamics, by means of a simple group additivity scheme and some common structural properties of globular proteins. The specific polar and nonpolar contributions to the heat capacity change are determined by analyzing the transfer of several different series of organic compounds to water. Additionally, we derive a general relationship for the evaluation of the fraction of accessible surface area buried in the protein interior, whose knowledge is necessary because only the groups that contact water on unfolding contribute to the heat capacity change. The model, despite its simplicity, works well, as the agreement between the calculated and experimental values of the denaturation heat capacity change is satisfactory for a large set of globular proteins.

Thermodynamics of protein stability: A family of ribonucleases

Ribonucleases constitute a ubiquitous superfamily of enzymes. Besides the well known hydrolytic activity against the RNAs, recently some of the proteins of this group, the so called RISBASE, have shown to possess a multiplicity of biological functions. In this article a short review is reported concerning systematic thermodynamic studies carried out at our laboratory mainly by means of differential scanning calorimetry on the well known ribonuclease A, RNase A, and its congeners. Among them, dimeric natural ribonuclease, BS-RNase, extracted from bull seminal plasma or vesicles, has shown very interesting features.

The effect ofpH on thermal stability of globular proteins

In this study we try to re-analyze thepH dependence of thermal stability of small globular proteins. From the thermodynamic point of view a long series of calorimetric and spectroscopic investigations has shown that the decreased stability in very acidic conditions can be ascribed to entropic effects. The same conclusion is reached, from a microscopic point of view, by assuming that a binding of protons on equal and noninteracting sites takes place as a consequence of unfolding process. By linking the conformational unfolding equilibrium to the proton binding equilibrium, a model is developed that is able to describe the dependence on thepH of the thermal denaturation processes of small globular protiens. The application of the model to hen lysozyme and T4 lysozyme correctly accounts for the experimental results.ZusammenfassungVorliegend versuchen wir eine Reanalyse der thermischen Stabilität von kleinen Globularproteinen. Unter thermodynamischen Aspekten zeigte eine lange Reihe von kalorimetrischen und spektroskopischen Untersuchungen, daß die verminderte Stabilität in sehr saurem Milieu Entropie-Effekten zugeschrieben werden kann. Zu der gleichen Schlußfolgerung gelangt man unter mikroskopischen Gesichtspunkten unter der Annahme, daß an entsprechenden Stellen, die keine Wechselwirkung eingehen, infolge eines Entfaltungsprozesses die Binding von Protonen stattfindet. Durch Verbindung des Konformations-Entfaltungsgleichgewichtes mit dem Protonenbindungsgleichgewicht wurde ein Modell entwickelt, welches sich zur Beschreibung derpH-Abhängigkeit des thermischen Denaturationsprozesses von kleinen Globularproteinen eignet. Die Anwendung des Modelles an Hühner-Lysozym und T4 Lysozym erklärt korrekt die experimentellen Ergebnisse.

Circular dichroism study of ribonuclease A mutants containing the minimal structural requirements for dimerization and swapping

Four residues Pro19. Leu28, Cys31 and Cys32 proved to be the minimal structural requirements in determining the dimeric structure and the N-terminal segment swapping of bovine seminal ribonuclease, BS-RNase. We analyzed the content of secondary and tertiary structures in RNase A, P-RNase A, PL-RNase A, MCAM-PLCC-RNase A and MCAM-BS-RNase, performing near and far-UV CD spectra. It results that the five proteins have very similar native conformations. Thermal denaturation at pH 5.0 of the proteins. studied by means of CD measurements. proved reversible and well represented by the two-state N<==>D transition model. Thermodynamic data are discussed in the light of the structural information available for RNase A and BS-RNase.

Linkage of proton binding to the thermal unfolding of Sso7d from the hyperthermophilic archaebacterium Sulfolobus solfataricus

In this study the pH dependence of the thermal stability of Sso7d from Sulfolobus solfataricus is analyzed. This small globular protein of 63 residues shows a very marked dependence of thermal stability on pH: the denaturation temperature passes from 65.2 degrees C at pH 2.5 to 97.9 degrees C at pH 4.5. Analysis of the data points out that the binding of at least two protons is coupled to the thermal unfolding. By linking the proton binding to the conformational unfolding equilibrium, a thermodynamic model, which is able to describe the dependence upon the solution pH of both the excess heat capacity function and the denaturation Gibbs energy change for Sso7d, is developed. The decreased stability in very acid conditions is due to the binding of two protons on identical and noninteracting sites of the unfolded state. Actually, such sites are two carboxyl groups possessing very low pKa values in the native structure, probably involved in salt-bridges on the protein surface.

On the Nature of the Temperature-Induced Transition From the Molten Globule to the Unfolded State of Globular Proteins

In this paper we try to perform a thermodynamic analysis of the temperature-induced transition from the molten globule to the unfolded state of globular proteins. A series of calorimetric investigations showed that this process is not associated with an excess heat capacity absorption peak, and cannot be regarded as a first-order phase transition. This result contrasts with the well-established conclusion that the thermal unfolding of the native tertiary structure of globular proteins is a first-order phase transition. First, the theoretical approach developed by Ikegami is outlined to emphasize that a second-order or gradual transition induced by temperature is expected for globular proteins when the various secondary structure elements do not interact cooperatively. Secondly, a simple thermodynamic model is presented which, taking into account the independence of the secondary structure elements among each other, is able to rationalize the shape of the experimental DSC profiles.

The enthalpy-convergence temperature for the dissolution into water of solid α-amino acids

Abstract In this paper it is pointed out that there is a convergence temperature for the enthalpy change associated with the dissolution process into water of four solid α-amino acids: glycine, dl -α-alanine, dl -α-aminobutyric acid and dl -α-norvaline. It is important that the value of the convergence temperature for α-amino acids, T ∗ H = 91.2 ± 6.3° C is very close to that determined for the denaturation of small globular proteins, T ∗ H = 100 ± 6° C . This result is analysed thoroughly in order to obtain information about the energetics of the protein denaturation process. The analysis points out that there is an energy penalty, due to the dehydration and burial of polar groups, that tends to counterbalance the energy gain due to the formation of polar and dispersive interactions in the close-packed interior of globular proteins.

The extra-stability of thermophilic globular proteins: a thermodynamic approach☆

Abstract In this paper a general thermodynamic analysis of the Gibbs energy change associated with the two-state denaturation process of small globular proteins is presented. The proposed “parabolic approximation” to the Gibbs energy change, apart from an analytical relationship for calculating the hot and cold denaturation temperatures, emphasizes the limiting thermodynamic mechanisms that a globular protein can exploit to increase its thermostability. These mechanisms are critically discussed, by stressing the fact that, actually, they are mutually dependent, due to the strong temperature-dependence of denaturation enthalpy and entropy changes.

Thermodynamic Stability of Ribonuclease B

The thermodynamic stability of pancreatic ribonuclease B (RNase B), which possesses identical protein structure of pancreatic ribonuclease A (RNase A), but differs by the presence of a carbohydrate chain attached to Asn 34, was studied by means of differential scanning calorimetry (DSC) at different pH conditions. The comparison between the two proteins has shown a little but significant stabilization of RNase B with respect to the unglycosylated one at pH values higher than 7.0. The thermodynamic analysis reveals the carbohydrate moiety to have a small stabilization effect of 3 kJ mol–1 at pH 8.0 and 63°C on the protein.

On the pH dependence of thermodynamic stability of α-amylase inhibitor tendamistat

In this study the pH dependence of the thermodynamic stability of tendamistat is analyzed. This small globular protein of 74 residues shows a very marked dependence of thermal stability on pH: the denaturation temperature increases from 68.9°C at pH 2.0 to 93.2°C at pH 5.0, and then decreases to 77.8°C at pH 8.0. Analysis of the data indicates that the binding of two protons is coupled to the thermal unfolding at pH values below 4.0, whereas one proton is released by the protein at pH values above 5.0. By linking the proton binding to the conformational unfolding equilibrium, a thermodynamic model, which is able to describe the dependence upon the solution pH of the denaturation Gibbs energy change for tendamistat, is developed.