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.

[Lysozyme Stabilization under High Pressure: Differential Scanning Microcalorimetry].

The heat denaturation of lysozyme has been studied by high-pressure differential scanning microcalorimetry. It has been demonstrated that an increase in pressure has different influence on denaturation temperature and enthalpy at different pH values. It has been established that the pressure increase has no appreciable effect on the transition cooperativity. The experimental data have been analyzed using an equilibrium model of transition between two states. Partial molar volume changes accompanying the denaturation as well as isothermal compressibility and thermal expansibility coefficients have been assessed. In contrast to the denaturation of most globular proteins, the lysozyme denaturation under conditions of the experiment was accompanied by positive volume changes. Possible reasons for this unusual behavior have been discussed.

High-pressure differential scanning microcalorimeter.

A differential scanning microcalorimeter for studying thermotropic conformational transitions of biopolymers at high pressure has been designed. The calorimeter allows taking measurements of partial heat capacity of biopolymer solutions vs. temperature at pressures up to 3000 atm. The principles of operation of the device, methods of its calibration, as well as possible applications are discussed.

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

Possible effects of high pressure on heat denaturation of biopolymers have been analyzed. The study was made using an equilibrium model of transition between two states. Equations 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 is demonstrated that depending on the increment (jump) of the partial expansion coefficient and the change of the transition temperature, the enthalpy of transition can both grow with the pressure growth or decrease. It is also .shown that when the pressure grows, the changes in the volume and enthalpy under denaturation cannot change their signs.

[Spin isomeric selectivity of water molecules upon DNA hydration].

Four-wave photon coherent scattering of laser emission was used to study the influence of DNA on the content of quasi-free ortho and para isomers of water molecules in its aqueous solution. It was shown that the concentration of quasi-free molecules that form the rotational spectrum of spin isomers increases considerably in the hydrational shell of the DNA molecule as compared to pure water. The increase in the concentration of spin isomers occurs disproportionally. In the presence of DNA, the intensity of the rotational spectrum of orthoisomers is on the average much greater than that of para isomers. It was also demonstrated that the character of hydration and the ortho/para ratio change noticeably upon DNA denaturation, which may be evidence of changes in preferable solvation of DNA during its denaturation. The data obtained allowed us to assume that the stability of different biologically important states of macromolecules can be changed by varying the relative concentration of water spin isomers in solution.

[Structural changes in wild-type Cry3A delta-endotoxin and its mutants in ethyl alcohol solutions at pH 2-2.5].

The methods of circular dichroism, scanning microcalorimetry, electron microscopy, and proteolysis were used to study the ability of wild-type Cry3A-delta-endotoxin and three mutant toxins with cysteine substitutions in helices alpha3 and alpha4 (domain I) to form oligomeric structures in acidic alcohol solutions that reproduce the premembrane environment. At pH 2-2.2 and 20% ethanol, the mutant toxins with single substitutions E132C (alpha3) and E160C (alpha4), as well as the double mutant E132C/S160C with a cysteine bridge connecting helices alpha3 and alpha4, form short linear oligomers specific for Cry3A with a high content of the beta-structure and enhanced sensitivity to proteolysis with pepsin. The data obtained show that the formation of oligomeric structures of this type does not require any divergence of helices alpha3 and alpha4 in domain I of the Cry3A toxin. It has been demonstrated that, at higher pH values in 20% solution of ethanol, the proteins studied are in a metastable state, and their ability to form oligomeric structures depends on temperature.

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.

[Destabilization and stabilization of poly(A).poly(U) structure by platinum(II) compounds].

On the basis of molecular biophysics, a methodology for the analysis of intramolecular structural order of the polynucleotide duplex poly(A).poly(U) has been developed. It was shown that the combination of circular dichroism spectroscopy with differential scanning calorimetry is an optimal approach, which ensures the screening of a wide set of substances and interaction conditions and the choice of compound(s) that can stabilize the structure and increase the biological activity of this duplex. The study is aimed at obtaining a new and highly active antiviral remedy.

High pressure effect on the main transition from the ripple gel P'beta phase to the liquid crystal (Lalpha) phase in dipalmitoylphosphatidylcholine. Microcalorimetric study.

Scanning microcalorimetry has been used to study the high pressure effect on the main transition from the ripple gel P'(beta) phase to the liquid crystal (L(alpha)) phase in DPPC (dipalmitoylphosphatidylcholine). It has been demonstrated that an increase of the pressure by 200 MPa shifts the transition to higher temperatures by 36.4 degrees. The pressure increase does not affect the cooperativity of transition but reduces noticeably its enthalpy. The changes of the molar partial volume, isothermal compressibility as well as volume thermal expansibility during transition in DPPC suspension have been estimated. It has been shown that monovalent ions (Na(+), Cl(-)) in solution slightly affect the main thermodynamic parameters of the transition. Calcium ions significantly decrease distinction in compressibility and thermal expansibility between liquid-crystal and ripple gel phases of lipid suspension, which in its turn reflects less difference in their volume fluctuations.

Spin-dependent absorption of water molecules.

The effects of spin state of water molecules on its absorption on lyophilized DNA, lysozyme and some inorganic sorbents were studied. It was shown that the absorption rates of ortho and para water from vapor differ noticeably. The para isomer binding with preparations is distinctly faster than that of the ortho isomer in all cases. Clear-cut distinction in the sorption kinetics is determined by the difference in quantum statistics for spin isomers, which in its turn can give rise to remarkable differences in physico-chemical properties of ortho and para water. This finding opens a wide field of activity in studying fundamental and applied problems relating to the role of the spin state of water molecules in physics, chemistry, biology and medicine.

[Structural changes in Cry3A delta-endotoxin from Bacillus thuringiensis var. Tenebrionis in alcohol solutions at pH 2.0].

Conformational changes in Cry3A delta-endotoxin caused by three different alcohols (ethanol, butanol, and isopropanol) were studied using the methods of circular dichroism, scanning microcalorimetry, and electron miscroscopy. It was shown that, in addition to the standard decrease in the native structure stability, the alcohols can cause a conformational transition that results in a sharp increase in the beta-structure content and a change in the environment of aromatic residues. The conformational transition is accompanied by intermolecular association, which leads to the appearance of oligomers in the form of short filaments. When the alcohols were removed, the oligomers dissociated again into monomers, but it is likely that the native structure either is not restored or is restored only in a small portion of molecules. The oligomer structure is rather cooperative, and its thermostability is higher than that of the initial structure. The disruption of this structure upon heating, observed as a heat absorption peak, is reversible.

Shift of fibril-forming ability of the designed alpha-helical coiled-coil peptides into the physiological pH region.

Recently, we designed a short alpha-helical fibril-forming peptide (alphaFFP) that can form alpha-helical nanofibrils at acid pH. The non-physiological conditions of the fibril formation hamper biomedical application of alphaFFP. It was hypothesized that electrostatic repulsion between glutamic acid residues present at positions (g) of the alphaFFP coiled-coil sequence prevent the fibrillogenesis at neutral pH, while their protonation below pH 5.5 triggers axial growth of the fibril. To test this hypothesis, we synthesized alphaFFPs where all glutamic acid residues were substituted by glutamines or serines. The electron microscopy study confirmed that the modified alphaFFPs form nanofibrils in a wider range of pH (2.5-11). Circular dichroism spectroscopy, sedimentation, diffusion and differential scanning calorimetry showed that the fibrils are alpha-helical and have elongated and highly stable cooperative tertiary structures. This work leads to a better understanding of interactions that control the fibrillogenesis of the alphaFFPs and opens opportunities for their biomedical application.

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.

Structure of Cry3A delta-endotoxin within phospholipid membranes.

Interaction of delta-endotoxin and its proteolytic fragments with phospholipid vesicles was studied using electron microscopy, scanning microcalorimetry, and limited proteolysis. It was shown that native protein destroys liposomes. The removal of 4 N-terminal alpha-helices and the extreme 56 C-terminal amino acid residues did not affect this ability. The results obtained by limited proteolysis of delta-endotoxin bound to lipid vesicles show essential conformational changes in three or four N-terminal helices and in the C-terminal region. The calorimetric method used in this study provides a unique possibility for the validation of existing models of protein binding and for a more accurate determination of the regions where conformational changes take place. It was found that the binding of the protein to model liposomes does not alter its structure in the regions starting with the fourth alpha-helix of domain I. This can be concluded from the fact that the activation energy of denaturation of the protein remains unchanged upon its binding to the phospholipid membranes. A new structural model has been proposed which agrees with the data obtained.

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.

Transition state of the rate-limiting step of heat denaturation of Cry3A delta-endotoxin.

Heat denaturation of Cry3A delta-endotoxin from Bacillus thuringiensis var. tenebrionis and its 55 kDa fragment was studied by differential scanning microcalorimetry at low pH. Analysis of the calorimetric data has shown that denaturation of Cry3A delta-endotoxin is a nonequilibrium process at heating rates from 0. 125 to 2 K/min. This means that the stability of delta-endotoxin (the apparent temperature of denaturation Tm) under these conditions is under kinetic control rather than under thermodynamic control. It has been shown that heat denaturation of this protein is a one-step kinetic process. The enthalpy of the process and its activation energy were measured as functions of temperature. The data obtained allow confirmation of the fact that the conformation of delta-endotoxin at the transition state only slightly differs from its native conformation with respect to compactness and extent of hydration. The comparison of the activation energy for intact delta-endotoxin and the 55 kDa fragment showed that the transition of the molecule to a transition state does not cause any changes in the conformation of three N-terminal alpha-helices. Complete removal of the N-terminal domain of delta-endotoxin and 40 amino acids from the C-terminus beta-sheet domain III causes an irreversible loss of the tertiary structure. Thus, during protein folding the nucleation core determining protein stability does not involve its three initial alpha-helices but may include the remaining alpha-helices of the N-terminal domain. The functional significance of peculiarities of structure arrangement of the delta-endotoxin molecule is discussed.

[Influence of kinetic factors on heat denaturation and renaturation of biopolymers]. / Vliianie kineticheskikh faktorov na teplovuiu denaturatsiiu i renaturatsiiu biopolimerov.

Possible influence of experimental nonequilibrium conditions on heat denaturation and renaturation of biopolymers has been studied. The analysis has been made using a standard kinetic model of transition between two states. Regularities are revealed which determine the position and shape of the heat absorption peak in calorimetric experiments on direct and reverse scanning (heating and cooling). The evaluation formulas for obtaining kinetic information from such experiments are given and their application is discussed. The results obtained can be useful in analysis of real calorimetric curves. It is shown that upon heating and cooling in nonequilibrium conditions the behavior of the system can differ qualitatively. The above difference takes place when the renaturation rate decreases on lowering the temperature. In this case, heating under nonequilibrium conditions leads only to a shift of denaturation transition to the higher temperatures while nonequilibrium cooling may result in complete or partial irreversibility.

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.