Sergey A. Potekhin

Microcalorimetric studies of conformational transitions of ferricytochrome c in acidic solution

The conformational transitions of ferricytochrome c in acidic solutions with different NaCl concentrations have been studied by scanning and isothermal microcalorimetry. It is shown that ferricytochrome c adopts three different forms which are realized under the considered conditions: native, denatured (unfolded) and a compact native-like form with unique tertiary structure. The thermodynamic parameters of the corresponding transitions have been measured and the changes in the number of bound ligands (H+ and Cl-), accompanying these transitions, have been determined by analyzing the temperature, pH and ionic strength dependence of these parameters.

High pressure stabilization of collagen structure

Scanning microcalorimetry has been used to study heat denaturation of small es, Cyrillicollagen at high pressure. It has been demonstrated that an increase of the pressure by 200 MPa enlarges the structure stability by 6.8 degrees C. The pressure increase does not affect the cooperativity of transition and only slightly decreases its enthalpy. The changes of the partial specific volume of collagen as well as its isothermal compressibility and thermal expansibility during transition have been estimated. In contrast to denaturation of most globular proteins, denaturation of collagen proceeds with an increase of the partial specific volume at ambient pressure. Moreover, the absolute value of the collagen volume increment is distinctly greater than this in most globular proteins. The volume increment diminishes when the pressure increases due to the difference in the compressibility for folded and unfolded states. This effect is compensated to some extent by the differences in heat expansion. The volume increment alters its sign when the pressure reaches about 324+/-20 MPa and the temperature of denaturation grows to 49.7 degrees C. Further pressure growing destabilizes the collagen structure.

DIFFERENTIAL SCANNING MICROCALORIMETER SCAL-1

The paper describes the design and the principle of operation of the differential scanning microcalorimeter SCAL-1. This instrument serves to measure thermal parameters of protein or biopolymer solutions of low concentration (from0.1 mg ml−1) in the temperature range from −10 to 130°C using different rates. A distinctive feature of the microcalorimeter is that its calorimetric cell is made of glass, it is highly sensitive and measuring results can be easily and reliably reproduced.

On the stabilizing action of protein denaturants: acetonitrile effect on stability of lysozyme in aqueous solutions.

Stability of hen lysozyme in the presence of acetonitrile (MeCN) at different pH values of the medium was studied by scanning microcalorimetry with a special emphasis on determination of reliable values of the denaturational heat capacity change. It was found that the temperature of denaturation decreases on addition of MeCN. However, the free energy extrapolation showed that below room temperature the thermodynamic stability increases at low concentrations of MeCN in spite of the general destabilizing effect at higher concentrations and temperatures. Charge-induced contribution to this stabilization was shown to be negligible (no pH-dependence was found); therefore, the most probable cause for the phenomenon is an increase of hydrophobic interactions at low temperatures in aqueous solutions containing small amounts of the organic additive. The difference in preferential solvation of native and denatured states of lysozyme was calculated from the stabilization free energy data. It was found that the change in preferential solvation strongly depends on the temperature in the water-rich region. At the higher MeCN content this dependence decreases until, at 0.06 mole fractions of MeCN, the difference in the preferential solvation between native and denatured lysozyme becomes independent of the temperature over a range of 60 K. The importance of taking into account non-ideality of a mixed solution, when analyzing preferential solvation phenomena was emphasized.

Folding Under Inequilibrium Conditions as a Possible Reason for Partial Irreversibility of Heat-Denatured Proteins: Computer Simulation Study

Using computer simulations we have studied possible effects of heating and cooling at different scan rates on unfolding and refolding of macromolecules. We have shown that even the simplest two-state reversible transition can behave irreversibly when an unfavorable combination of cooling rate, relaxation time and activation energy of refolding occurs. On the basis of this finding we suppose that apparent irreversibility of some proteins denatured by heat may result from slow relaxation on cooling rather than thermodynamic instability and/or irreversible alterations of the polypeptide chain. Using this kinetic reversible two-state model, we estimated the effects of the scan rate and kinetic parameters of the macromolecule on its unfolding-refolding process. A few recommendations are suggested on how to reach maximal possible recovery after denaturation if refolding appears to be under kinetic control.

STRUCTURAL PROPERTIES OF ALPHA -FETOPROTEIN FROM HUMAN CORD SERUM : THE PROTEIN MOLECULE AT LOW PH POSSESSES ALL THE PROPERTIES OF THE MOLTEN GLOBULE

Structural studies of α‐fetoprotein (AFP) from human cord serum have shown that a decrease in pH to 3.1 leads to a considerable conformational rearrangement of the protein molecule. The acid form of AFP belongs to the class of denatured conformations and fulfills all the requirements of the molten globule state. The possible functional role of such a transformation is discussed.

Thermodynamic Analysis of Two State Transitions under High Pressure. Theoretical Consideration

The possible effects of high pressure on the heat denaturation of biopolymers have been analyzed. The study was performed using an equilibrium model of the transition between two states. The equations that were used to determine the dependence of thermodynamic parameters of transition (enthalpy, entropy, and transition temperature) on pressure were formulated. Pressure may have both stabilizing and destabilizing effects on the macromolecule structure depending on the change in the volume upon transition. The dependence of the transition temperature on pressure cannot have local extremums. It was demonstrated that, depending on the increment (jump) of the partial expansion coefficient and the change of the transition temperature, the enthalpy of the transition can either increase or decrease with pressure. It was also shown that when the pressure increases the changes in the volume and enthalpy under denaturation cannot change their signs.

Mutation Gln54Leu of the conserved polar residue in the interfacial coiled coil position (d) results in significant stabilization of the original structure of the COMP pentamerization domain

The assembly domain of cartilage oligomeric matrix protein (COMP) forms an α-helical coiled coil homopentamer with a conserved polar glutamine in the interior (d) position. We substituted Gln54 for apolar Leu in the recombinant fragment of the rat COMP domain. Biochemical studies and circular dichroism (CD) spectroscopy showed that the mutant, similarly to the wild-type (w.t.) peptide, forms spontaneously an α-helical pentamer. Thermal transitions of the w.t. and mutant pentamers were analyzed by CD spectroscopy and differential scanning calorimetry. The Gln54Leu mutation increased the thermal stability of the pentamer with reduced disulfide bonds from 73°C to 104°C. The denaturation of the disulfide bonded w.t. pentamer was observed at 108°C while the mutant pentamer cannot be denatured up to 120°C (the apparatus limit). Thus, by Gln54Leu mutation we found a way to significantly stabilize the coiled coil pentamer, making this peptide even more attractive as an oligomerization tool for various biotechnological applications.

High-Pressure Scanning Microcalorimetry – A New Method for Studying Conformational and Phase Transitions

The development of high-pressure scanning microcalorimetry and the first results studying transitions in proteins, lipids, and model polymers are reviewed. Special attention is given to changes (increments) in volume parameters upon transitions as well as the nature of these changes. It is demonstrated that the use of the model of compound transfer reaction in its purest form for assessment of denaturation volume effects failed due to serious difficulties.

Dependence on acyl chain length of energy and volume parameters of the gel to liquid-crystalline transition of 1,2–diacylphosphatidylcholines. Theoretical consideration

The effect of acyl chain length on energy and volume parameters of gel to liquid-crystal transitions in phospholipids is analyzed. It is demonstrated that simple structural and thermodynamic considerations allow predicting some thermodynamic and volume characteristics of transitions and their dependencies on the acyl chains length.