[Experience in simulating the structural and dynamic features of small proteins using table supercomputers].

The results of theoretical studies of the structural and dynamic features of peptides and small proteins have been presented that were carried out by quantum chemical and molecular dynamics methods in high-performance graphic stations, "table supercomputers", using distributed calculations by the CUDA technology.

Side chain dynamics and alternative hydrogen bonding in the mechanism of protein thermostabilization.

To elucidate the mechanism of protein thermostabilization, the thermodynamic properties of small monomeric proteins from mesophilic and thermophilic organisms have been analyzed. Molecular dynamics simulations were employed in the study of dynamic features of charged and polar side chains of amino acid residues. The basic conclusion has been made: surface charged and polar side chains with high conformational mobility can form alternative hydrogen bonded (H-bonded) donor-acceptor pairs. The correlation between the quantitative content of alternative H-bonds per residue and the temperature of maximal thermostability of proteins has been found. The proposed mechanism of protein thermostabilization suggests continuous disruption of the primary H-bonds and formation of alternative ones, which maintain constant the enthalpy value in the native state and prevent a rapid increase of the conformational entropy with the rising temperature. The analysis of the results show that the more residues located in the N- and C-terminal regions and in the extended loops that are capable of forming alternative longer-range H-bonded pairs, the higher the protein thermostability.

Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus.

The temperature adaptation of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus (Pf PCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The Pf PCP exhibited maximal catalytic activity at 90-95 degrees C and its activity was higher in the temperature range 30-100 degrees C than its counterpart from the mesophilic Bacillus amyloliquefaciens (BaPCP). Thermal stability was monitored by differential scanning calorimetry (DSC). Two clearly separated peaks appeared on the DSC curves for Pf PCP at alkaline and acidic pH. Using the oxidized Pf PCP and two mutant proteins (Pf C188S and Pf C142/188S), it was found that the peaks on the high and low temperature sides of the DSC curve of Pf PCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of intersubunit disulfide bridges. The denaturation temperature (Td) of Pf PCP with intersubunit disulfide bridges was higher by 53 degrees C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric Pf C142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of Pf PCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The Td of Pf C142/188S decreased as the enzyme became dissociated, but the monomeric form of the protein was still folded at pH 2.5, although BaPCP was completely denatured at acidic pH. These results indicate that subunit interaction plays an important role in stabilizing PCP from P. furiosus in addition to the intrinsic enhanced stability of its monomer.

Thermodynamics of the temperature-induced unfolding of globular proteins.

The heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature-induced unfolding of 11 globular proteins of known three-dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent-accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 degrees C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of non-polar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature-induced unfolding.

Thermodynamic properties of globular proteins and the principle of stabilization of their native structure.

A semi-empirical method has been used to estimate the thermodynamic parameters of hydration of buried surface areas of ribonuclease S, lysozyme and myoglobin from the model of complete unfolding according to Ooi et al. ((1987) Proc. Natl. Acad. Sci. USA 84, 3086-3090). The buried surface area of proteins is considered as the difference between the accessible surface area of native protein and the completely extended polypeptide chain according to Lee and Richards ((1971) J. Mol. Biol. 55, 379-400). The contributions of nonpolar and polar protein groups to the general value of Gibbs energy, enthalpy, entropy and heat capacity of hydration have been determined. The obtained results on the thermodynamic behavior of proteins in the process of complete unfolding are in good agreement with the results of microcalorimetric studies of thermal denaturation.

Heat capacity and conformation of proteins in the denatured state.

Heat capacity, intrinsic viscosity and ellipticity of a number of globular proteins (pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, myoglobin and cytochrome c) and a fibrillar protein (collagen) in various states (native, denatured, with and without disulfide crosslinks or a heme) have been studied experimentally over a broad range of temperatures. It is shown that the partial heat capacity of denatured protein significantly exceeds the heat capacity of native protein, especially in the case of globular proteins, and is close to the value calculated for an extended polypeptide chain from the known heat capacities of individual amino acid residues. The significant residual structure that appears at room temperature in the denatured states of some globular proteins (e.g. myoglobin and lysozyme) at neutral pH results in a slight decrease of the heat capacity, probably due to partial screening of the protein non-polar groups from water. The heat capacity of the unfolded state increases asymptotically, approaching a constant value at about 100 degrees C. The temperature dependence of the heat capacity of the native state, which can be determined over a much shorter range of temperature than that of the denatured state and, correspondingly, is less certain, appears to be linear up to 80 degrees C. Therefore, the denaturational heat capacity increment seems to be temperature-dependent and is likely to decrease to zero at about 140 degrees C.

Cooperativity of the alpha beta-protomer structure in Na+,K+-ATPase functioning. A scanning microcalorimetry study.

Heat denaturation of the free and ligand-bound forms of purified Na+,K+-ATPase from pig kidney is studied with the scanning microcalorimetry technique. A single two-state transition is observed during denaturation of the free enzyme, the molar concentration of the cooperatively melting units being equal to the concentration of alpha beta-protomers (Mr approximately equal to 140 000). Upon interaction of the enzyme with phosphate, Mg2+, and strophanthidin, but not with Na+, the cooperativity of the protomer unfolding is lost, and the protein stabilization enthalpy becomes approximately equal to 230 kJ/mol higher. The data suggest that in a functionally active enzyme form, the alpha beta-protomers possess a rigid structure with tight association of their subunits and domains, this structural rigidity is essential for the Na+,K+-ATPase functioning and there is a unique non-active conformation of the enzyme which may play an important role in its in vivo regulation.

Multidomain of flagellin.

Scanning microcalorimetric and circular dichroism studies of the normal and mutant flagellins of Salmonella suggest that they have a multidomain structure in common. Flagellin polymers (flagella) are depolymerized irreversibly into monomers as the temperature is raised, and the monomers undergo denaturation reversibly when cooled and heated again. The calorimetric enthalpy of this reversible process is twice as large as the van't Hoff enthalpy, suggesting that flagellin monomers contain two co-operative regions that melt independently at the same temperature. In all flagellin specimens examined, the ellipticity at the same temperature. In all flagellin specimens examined, the ellipticity at 222 nm of polymers at room temperature is 1.6 times as large as that of monomers, and the dependence of ellipticity on temperature takes place in the same temperature intervals in which calorimetric effects take place. From these results, we propose that flagellin molecules consist of several domains, two of which are distinctly structured in monomers at room temperature, while the others acquire more regular structures during polymerization.

[Calorimetric study of heat denaturation of toxins]. / Kalorimetricheskoe issledovanie teplovoi denaturatsii toksinov.

A calorimetric study of reversible heat denaturation of cytotoxin I, neurotoxins I and II in aqueous solution has been carried out. All of them are low molecular proteins from snake venom. Thermodynamic parameters of the transition of the toxins from native to denatured state were determined. Temperature dependences of a specific enthalpy delta dH of the transition were found. It was shown, that upon denaturation the changes in the value of partial heat capacity delta dCp for each of the toxins were constant and did not depend on medium conditions, i.e. composition of a solvent, pH and temperature of the transition. The results of the calorimetric study of the toxins are discussed along with structural peculiarities of low molecular weight proteins (less than 10 000 D) characterized by the amount of van der Waals' interactions between non-polar groups and intramolecular hydrogen bonds.

Calorimetric study of tryptophan synthase alpha-subunit and two mutant proteins.

Heat-denaturation of tryptophan synthase alpha-subunit from E. coli and two mutant proteins (Glu 49 leads to Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20 degrees, Cp20, has been found to be (0.43 +/- 0.02) cal . k-1 . g-1, the unfolding heat capacity change, delta dCp, (0.10 +/- 0.01) cal . K-1 . g-1, and the unfolding enthalpy value extrapolated to 110 degrees, delta dh110, (9.3 +/- 0.5) cal . g-1 for the three proteins. The value of Cp20 was larger than those found for "fully compact protein" and that of delta dh110 was smaller. Unfolding Gibbs energy, delta dG at 25 degrees for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal . mol-1 at pH 9.3, respectively. Unfolding enthalpy, delta dH, of the three proteins seemed to be the same and equal to (23.2 +/- 1.2) kcal . mol-1 at 25 degrees. As a consequence of the same value of delta dH and the different value in delta dG, substantial differences in unfolding entropy, delta dS, were found for the three proteins. The values of delta dG for the three proteins at 25 degrees coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.

Studies on the structure of protein L7/L12 from Escherichia coli ribosomes.

Circular dichroism, infrared and proton magnetic resonance spectroscopy as well as microcalorimetry methods were used to investigate the intact proteins L7/L12 in solution and their different derivatives (L7 with oxidized residues of methionine, fragments 27--120, 1--73 and 74--120)- On the basis of the data obtained the following conclusions have been drawn: (a) there is no beta structure in the protein L7, (B) the N-terminal region of L7 forms a long alpha helix (c) the Phe-30 residue within the N-terminal region of L7 takes part in the dimerization, (d) the C-terminal of L7 is globular and (e) the Phe-54 residue is included in the hydrophobic core of the globular C-terminal region.

Thermally induced conformational transitions of Bence-Jones protein IVA and its proteolytic fragments.

The thermally induced conformational transitions of the kappa-type Bence-Jones protein IVA and its proteolytic fragments (variable and constant halves) were studied by differential adiabatic scanning microcalorimetry, circular dichroism and thermal differential spectroscopy. The striking feature of the results is the good agreement between the experimental heat of thermal denaturation of intact Bence-Jones protein and the heat calculated from the individual variable and constant halves. The results suggested that the variable and constant halves have independent secondary and tertiary structures. It is likely that in the intact light chain, the variable and constant domains have weak non-covalent interactions between themselves. It was shown that at pH values from 7.4 to 2.0 the variable halves exist in the dimeric form. Evidence was obtained that two relatively independent regions of strong non-covalent interactions stabilize the dimers of variable and constant domains.