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P L Privalov

Publications and source records attributed to P L Privalov.

At least 19 recordsLinked to original sources
PubMed2001

Contribution of translational and rotational motions to molecular association in aqueous solution.

Much uncertainty and controversy exist regarding the estimation of the enthalpy, entropy, and free energy of overall translational and rotational motions of solute molecules in aqueous solutions, quantities that are crucial to the understanding of molecular association/recognition processes and structure-based drug design. A critique of the literature on this topic is given that leads to a classification of the various views. The major stumbling block to experimentally determining the translational/rotational enthalpy and entropy is the elimination of vibrational perturbations from the measured effects. A solution to this problem, based on a combination of energy equi-partition and enthalpy-entropy compensation, is proposed and subjected to verification. This method is then applied to analyze experimental data on the dissociation/unfolding of dimeric proteins. For one translational/rotational unit at 1 M standard state in aqueous solution, the results for enthalpy (H degrees (tr)), entropy (S degrees (tr)), and free energy (G degrees (tr)) are H (degrees) (tr) = 4.5 +/- 1.5RT, S (degrees) (tr) = 5 +/- 4R, and G (degrees) (tr) = 0 +/- 5RT. Therefore, the overall translational and rotational motions make negligible contribution to binding affinity (free energy) in aqueous solutions at 1 M standard state.

Bacterial Proteins↗
PubMed2000

A calorimetric study of the influence of calcium on the stability of bovine alpha-lactalbumin.

Bovine alpha-lactalbumin has been studied by differential scanning calorimetry with various concentrations of calcium to elucidate the effect of this ligand on its thermal properties. In the presence of excess calcium, alpha-lactalbumin unfolds upon heating with a single heat-absorption peak and a significant increase of heat capacity. Analysis of the observed heat effect shows that this temperature-induced process closely approximates a two-state transition. The transition temperature increases in proportion with the logarithm of the calcium concentration, which results in an increase in the transition enthalpy as expected from the observed heat capacity increment of denaturation. As the total concentration of free calcium in solution is decreased below that of the proteins, there are two temperature-induced heat absorption peaks whose relative area depends on the calcium concentration, such that further decrease of calcium concentration results in a increase of the low-temperature peak and a decrease of the high-temperature one. The high-temperature peak occurs at the same temperature as the unfolding of the holo-protein, while the low-temperature peak is within the temperature range associated with the unfolding of the apo-protein. Statistical thermodynamic modeling of this process shows that the bimodal character of the thermal denaturation of bovine alpha-lactalbumin at non-saturated calcium concentrations is due to a high affinity of Ca2+ for alpha-lactalbumin and a low rate of calcium exchange between the holo- and apo-forms of this protein. Using calorimetric data, the calcium-binding constant for alpha-lactalbumin has been determined to be 2.9 x 10(8) M-1.

Animals↗
PubMed2000

Energetics of the specific binding interaction of the first three zinc fingers of the transcription factor TFIIIA with its cognate DNA sequence.

The energetics of the specific interaction of a protein fragment (zf1-3) containing the three N-terminal zinc fingers of the Xenopus laevis transcription factor TFIIIA with its cognate DNA sequence, contained in a 15 bp DNA duplex were studied using isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and fluorescence titration. The use of both ITC and DSC is necessary to provide values for the thermodynamic parameters that have been corrected for thermal fluctuations of the interacting molecules. In the temperature range from 13 degrees C to 45 degrees C (where all the binding reaction components are folded), formation of the complex is enthalpically driven with a negative heat capacity effect (DeltaC(p)). In this respect, the binding reaction of zf1-3 is similar to those of other proteins that bind in the major groove of DNA. It is dissimilar to the association reactions of proteins, however, that bind in the minor groove of DNA and that are driven by a dominating entropy factor. Comparison of the experimental values of DeltaH(ass) and DeltaC(p) with expected values of these parameters, calculated from the burial of polar and nonpolar molecular surfaces, indicates that the polar groups at the protein/DNA interface are not completely dehydrated upon formation of the complex. It also seems that the expected large positive entropy of dehydration upon forming the zfl-3/DNA complex ( approximately 1900 J * K(-1) * mol(-1)) cannot be balanced by the reduction in translational/rotational and configurational freedom of the protein to the level of the observed entropy of binding (38 J * K(-1) * mol(-1)). It is suggested that the additional negative entropy contribution comes from a damping of torsional motions in the DNA duplex.

Calorimetry, Differential Scanning↗
PubMed1999

The energetics of HMG box interactions with DNA: thermodynamic description of the target DNA duplexes.

The thermal properties and energetics of formation of 10, 12 and 16 bp DNA duplexes, specifically interacting with the HMG box of Sox-5, have been studied by isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). DSC studies show that the partial heat capacity of these short duplexes increases considerably prior to the cooperative process of strand separation. Direct extrapolation of the pre and post-transition heat capacity functions into the cooperative transition zone suggests that unfolding/dissociation of strands results in no apparent heat capacity increment. In contrast, ITC measurements show that the negative enthalpy of complementary strand association increases in magnitude with temperature rise, implying that strand association proceeds with significant decrease of heat capacity. Furthermore, the ITC-measured enthalpy of strand association is significantly smaller in magnitude than the enthalpy of cooperative unfolding measured by DSC. To resolve this paradox, the heat effects upon heating and cooling of the separate DNA strands have been measured by DSC. This showed that cooling of the strands from 100 degrees C to -10 degrees C proceeds with significant heat release associated with the formation of intra and inter-molecular interactions. When the enthalpy of residual structure in the strands and the temperature dependence of the heat capacity of the duplexes and of their unfolded strands have been taken into account, the ITC and DSC results are brought into agreement. The analysis shows that the considerable increase in heat capacity of the duplexes with temperature rise is due to increasing fluctuations of their structure (e.g. end fraying and twisting) and this effect obscures the heat capacity increment resulting from the cooperative separation of strands, which in fact amounts to 200(+/-40) JK(-1) (mol bp)(-1). Using this heat capacity increment, the averaged standard enthalpy, entropy and Gibbs energy of formation of fully folded duplexes from fully unfolded strands have been determined at 25 degrees C as -33(+/-2) kJ (mol bp)(-1), -93(+/-4) J K(-1) (mol bp)(-1) and -5.0(+/-0.5) kJ (mol bp)(-1), respectively.

Animals↗
PubMed1999

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5.

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.

Animals↗
PubMed1999

A calorimetric study of the folding-unfolding of an alpha-helix with covalently closed N and C-terminal loops.

The thermal melting of a dicyclic 29-residue peptide, having helix-stabilizing side-chain to side-chain covalent links at each terminal, has been studied by circular dichroism spectropolarimetry (CD) and differential scanning calorimetry (DSC). The CD spectra for this dicyclic peptide indicate that it is monomeric, almost fully alpha-helical at -10 degrees C, and undergoes a reversible transition from the folded to the disordered state with increasing temperature. The temperature dependencies of the ellipticity at 222 nm and the excess heat capacity measured calorimetrically are well fit by a two-state model, which indicates a cooperative melting transition that is complete within the temperature ranges of these experiments (from -10 degrees C to 100 degrees C). This allows a complete analysis of the thermodynamics of helix formation. The helix unfolding is found to proceed with a small positive heat-capacity increment, consistent with the solvation of some non-polar groups upon helix unfolding. It follows that the hydrogen bonds are not the only factors responsible for the formation of the alpha-helix, and that hydrophobic interactions are also playing a role in its stabilization. At 30 degrees C, the calorimetric enthalpy and entropy values are estimated to be 650(+/-50) cal mol(-1)and 2.0(+/-0.2) cal K(-1)mole(-1), respectively, per residue of this peptide. Comparison with the thermodynamic characteristics obtained for the unfolding of double-stranded alpha-helical coiled-coils shows that at that temperature the enthalpic contribution of non-polar groups to the stabilization of the alpha-helix is insignificant and the estimated transition enthalpy can be assigned to the hydrogen bonds. With increasing temperature, the increasing magnitude of the negative enthalpy of hydration of the exposed polar groups should decrease the helix-stabilizing enthalpy of the backbone hydrogen bonds. However, the helix-stabilizing negative entropy of hydration of these groups should also increase in magnitude with increasing temperature, offsetting this effect.

Amino Acid Sequence↗
PubMed1998

The energetics of HMG box interactions with DNA. Thermodynamic description of the box from mouse Sox-5.

The structural energetics of the HMG box from the DNA-binding protein mouse Sox-5 were examined calorimetrically. It was found that this box, notwithstanding its small size (molecular mass about 10 kDa), does not behave as a single cooperative unit and, on heating, the box reversibly unfolds in two separate stages. The first transition (tt approximately 34 degrees C) involves about 40% of the total enthalpy and the second (tt approximately 46 degrees C) the remainder. Both transitions proceed with significant heat capacity increment, showing that they are associated with the unfolding of two sub-domains having non-polar cores. According to heat capacity, ellipticity, fluorescence and NMR criteria, this HMG box is in a fully compact native state only below 5 degrees C. HMG boxes consist of two approximately orthogonal wings: the minor wing comprises helix 3 and its associated antiparallel N-terminal strand, whilst the major wing is composed of helices I and II. Analysis of the fluorescence and NMR spectra for this box obtained at different temperatures shows that the lower melting transition can be assigned to the minor wing and the upper transition to the major wing. Under physiological conditions (37 degrees C), the minor wing is considerably unfolded, whilst the major wing is essentially fully folded. DNA binding in vivo therefore involves refolding of the minor wing.

Animals↗
PubMed1997

The entropy cost of protein association.

The temperature induced unfolding/dissociation of the dimeric subtilisin inhibitor from Streptomyces and its mutant D83C having an S-S crosslink between the subunits has been studied calorimetrically. Comparison of the entropies measured at different concentrations of dimer showed that the entropy cost of crosslinking is small. Its value at the standard concentration of 1 M is of the order of -(5+/-4) cal/K.mol, i.e. it is more than one order of magnitude smaller than the values of translational entropies calculated on the base of statistical thermodynamics, using in particular the Sackur-Tetrode equation, and is close to the cratic entropy value suggested by classical mixing theory.

Bacterial Proteins↗
PubMed1997

Energetics of target peptide recognition by calmodulin: a calorimetric study.

Calmodulin is a small protein involved in the regulation of a wide variety of intracellular processes. The cooperative binding of Ca2+ to calmodulin's two Ca2+ binding domains induces conformational changes which allow calmodulin to activate specific target enzymes. The association of calmodulin with a peptide corresponding to the calmodulin binding site of rabbit smooth muscle myosin light chain kinase (smMLCKp) was studied using isothermal titration microcalorimetry. The dependence of the binding energetics on temperature, pH, Ca2+ concentration, and NaCl concentration were determined. It is found that the binding of calmodulin to smMLCKp proceeds with negative changes in enthalpy (deltaH), heat capacity (deltaCp), and entropy (deltaS) near room temperature, indicating that it is an enthalpically driven process that is entropically unfavorable. From these results it is concluded that the hydrophobic effect, an entropic effect which favors the removal of non-polar protein groups from water, is not a major driving force in calmodulin-smMLCKp recognition. Although a large number of non-polar side-chains are buried upon binding, these stabilize the complex primarily by forming tightly packed van der Waals interactions with one another. Binding at acidic pH was studied in order to assess the contribution of electrostatic interactions to binding. It is found that moving to acidic pH results in a large decrease in the Gibbs free energy of binding but no change in the enthalpy, indicating that electrostatic interactions contribute only entropically to the binding energetics. The accessible surface area and atomic packing density of the calmodulin-smMLCKp crystal structure are analyzed, and the results discussed in relation to the experimental data.

Amino Acid Sequence↗
PubMed1997

Heat capacities of protein functional groups.

Using a precise technique of scanning calorimetry the heat capacities of a series of carboxylic acids and their sodium salts, alcohols, and N-substituted amides have been measured from 5 to 100 degrees C. From these data, the partial molar heat capacities of CH2, CONH, COOH, and COONa groups have been determined. It is shown that the heat capacity of the CH(2) group in aqueous solution is independent of the type of compound used for its determination, is positive at low temperature, and is linearly decreasing in magnitude with an increase in temperature. In contrast, the heat capacities of COOH and COONa groups in aqueous solution are negative at room temperature and their magnitude non-linearly decreases with an increase in temperature. It appears that the partial heat capacity of CONH group in aqueous solution depends on the type of model compound used for its determination. These differences correlate with the difference in the water accessible surface area of atoms in the CONH group in different model compounds.

Journal Article↗
PubMed1997

Energetics of folding and DNA binding of the MAT alpha 2 homeodomain.

Homeodomains are a class of DNA-binding protein domains which play an important role in genetic regulation in eukaryotes. We have characterized the thermodynamics of folding and sequence-specific association with DNA of the MAT alpha 2 homeodomain of yeast. Using differential scanning and isothermal titration calorimetry, we measured the enthalpy, heat capacity, and Gibbs free energy changes of these processes. The protein-DNA interaction is enthalpically driven at physiological temperatures. DSC data on the process of melting the protein-DNA complex at different salt concentrations were dissected into its endothermic components, yielding the enthalpy change and dissociation constant of binding. A comparison of the circular dichroism spectra of the free and DNA-bound protein species revealed the formation of additional alpha-helical structure upon binding to DNA. We propose that the latter half of helix 3, the recognition helix, is substantially unfolded in the free protein under the conditions used, as has been observed with other homeodomains [Tsao, D. H. H., et al. (1994) Biochemistry 33, 15053-15060: Cox, M., et al. (1995) J. Biomol. NMR 5, 23-32]. Formation of protein structure is induced by DNA binding, and the energies measured for association therefore include a component due to folding.

Circular Dichroism↗
PubMed1996

Intermediate states in protein folding.

The efficiency of protein folding suggests that this process proceeds through some intermediate states, raising the possibility of experimental observation of incompletely folded states of proteins. However, critical analysis of the observed partly folded stable states of proteins shows that they present either misfolded forms obtained under conditions inappropriate for folding, or partially unfolded states that retain folded the subpart of these molecules. This retained part, which unfolds last and folds first in a unfolding/refolding experiment, has a definite tertiary structure maintained by specific long-range interactions and can be isolated by fragmentation. Therefore, it can be regarded as a definite domain of the protein molecule. Provided the polypeptide chain of a small single domain protein does not become trapped in a misfolded form, its folding proceeds very rapidly, with all intermediates being transient and extremely unstable.

Animals↗
PubMed1996

Investigation of electrostatic interactions in two-stranded coiled-coils through residue shuffling.

The effects of electrostatic interactions on the stability of coiled-coils were investigated using the strategy of shuffling the sequence without changing the overall content of amino acid residues in the peptides. Shuffling the sequence provides peptides with thermodynamically similar unfolded states. Therefore, the unfolded state can be used as a universal reference state in comparing the thermodynamic properties of the folded coiled-coil structure of the peptides, while varying the configuration of ionized groups, that is, changing the types and number of potential electrostatic interactions. The relative stabilities of these states were determined by monitoring the temperature-induced folding/unfolding of the peptides in solutions with different pH and ionic strength by circular dichroism spectroscopy and scanning microcalorimetry. It was found that, in solutions with low ionic strength, ionic pairs contribute significantly to the stability of the coiled-coil conformation. The stability increases with an increase in the number of ionized groups in the peptide upon changing pH from acidic to neutral. In contrast, in the solutions with high ionic strength, the coiled-coil becomes less stable at neutral pH than at acidic pH. Most surprisingly, the increase in Gibbs energy of stabilization of the coiled-coil state with increasing pH at low ionic strength proceeds with a decrease in the enthalpy and entropy of unfolding. This observation can be explained only by hydration of ionized groups upon unfolding of coiled-coils which is associated with significant negative enthalpy and entropy effects.

Amino Acid Sequence↗
PubMed1996

Ion pairs significantly stabilize coiled-coils in the absence of electrolyte.

We have used a synthetic coiled-coil peptide model system to address the long perplexing issue as to why coiled-coils are in general more stable at acidic pH than at neutral pH. Contrary to the above expectation, our results show that at low ionic strength (10 mM) the coiled-coil was much more stable at neutral pH than at acidic pH against both thermal and urea unfolding, indicating that the Lys(+)-Glu- ions pairs present around the coiled-coil interface at neutral pH contribute significantly to the stability of the coiled-coil. However, while the addition of NaCl had no significant effect on the coiled-coil stability at neutral pH, its stability at acidic pH increased dramatically. The cross-over point between the stability at acidic pH and neutral pH occurred at around 100 mM salt, above which the coiled-coil became more stable at acidic pH, in agreement with published results. Therefore, salt effect, rather than intrinsic property, such as carboxyl-carboxyl hydrogen bonding, causes this coiled-coil to become more stable at acidic pH. The preferential stabilizing effect of salt on the coiled-coil at acidic pH can be best explained in terms of the condensation of anions to the positively charged groups on the coiled-coil, the net density of which increases as glutamic acid residues become protonated in acidic pH.

Amino Acid Sequence↗
PubMed1996

On the entropy of protein folding.

The failure to appreciate that the hydration of polar groups is a major contribution to the entropy of protein unfolding has led to considerable underestimates for the loss of configurational freedom when a protein chain folds.

Bacterial Proteins↗
PubMed1996

Thermodynamic effects of mutations on the denaturation of T4 lysozyme.

We investigated the folding of substantially destabilized mutant forms of T4 lysozyme using differential scanning calorimetry and circular dichroism measurements. Three mutations in an alpha-helix in the protein's N-terminal region, the alanine insertion mutations S44[A] and K48[A], and the substitution A42K had previously been observed to result in unexpectedly low apparent enthalpy changes of melting, compared to a pseudo-wild-type reference protein. The pseudo-wild-type reference protein thermally unfolds in an essentially two-state manner. However, we found that the unfolding of the three mutant proteins has reduced cooperativity, which partially explains their lower apparent enthalpy changes. A three-state unfolding model including a discrete intermediate is necessary to describe the melting of the mutant proteins. The reduction in cooperativity must be considered for accurate calculation of the energy changes of folding. Unfolding in two stages reflects the underlying two-subdomain structure of the lysozyme protein family.

Bacteriophage T4↗
PubMed1996

Thermodynamics of denaturation of staphylococcal nuclease mutants: an intermediate state in protein folding.

A valuable approach to understanding the forces that maintain protein structure is to analyze the thermodynamic effects of mutations on protein folding. The folding process is most often described using an energetic model that assumes a two-state transition between the native and denatured states. However, some results obtained using this approach for mutants of the protein staphylococcal nuclease have contradicted expectations from our current understanding of protein energetics. The application of differential scanning calorimetry to a set of mutant nuclease proteins allowed us to measure directly the effects of mutations on the enthalpy and heat capacity changes of unfolding, as well as on the cooperativity. We found that most of these effects can be understood with a three-state model of folding including a distinct intermediate, but not with the two-state model. Use of a three-state instead of a two-state model leads to large differences in conclusions about the stability effects of some mutations, suggesting that reevaluation of the effects of mutations on this and other proteins may be necessary to achieve an accurate description of folding energetics. The two-state assumption commonly used in protein stability studies may be an oversimplification in many cases.

Micrococcal Nuclease↗