Thermodynamics and kinetics of a folded-folded' transition at valine-9 of a GCN4-like leucine zipper

Spin inversion transfer (SIT) NMR experiments are reported probing the thermodynamics and kinetics of interconversion of two folded forms of a GCN4-like leucine zipper near room temperature. The peptide is 13Calpha-labeled at position V9(a) and results are compared with prior findings for position L13(e). The SIT data are interpreted via a Bayesian analysis, yielding local values of T1a, T1b, kab, kba, and Keq as functions of temperature for the transition FaV9 right arrow over left arrow FbV9 between locally folded dimeric forms. Equilibrium constants, determined from relative spin counts at spin equilibrium, agree well with the ratios kab/kba from the dynamic SIT experiments. Thermodynamic and kinetic parameters are similar for V9(a) and L13(e), but not the same, confirming that the molecular conformational population is not two-state. The energetic parameters determined for both sites are examined, yielding conclusions that apply to both and are robust to uncertainties in the preexponential factor (kT/h) of the Eyring equation. These conclusions are 1) the activation free energy is substantial, requiring a sparsely populated transition state; 2) the transition state's enthalpy far exceeds that of either Fa or Fb; 3) the transition state's entropy far exceeds that of Fa, but is comparable to that of Fb; 4) "Arrhenius kinetics" characterize the temperature dependence of both kab and kba, indicating that the temperatures of slow interconversion are not below that of the glass transition. Any postulated free energy surface for these coiled coils must satisfy these constraints.     

Site-specific experiments on folding/unfolding of Jun coiled coils: thermodynamic and kinetic parameters from spin inversion transfer nuclear magnetic resonance at leucine-18

The 32-residue leucine zipper subsequence, called here Jun-lz, associates in benign media to form a parallel two-stranded coiled coil. Studies are reported of its thermal unfolding/folding transition by circular dichroism (CD) on samples of natural isotopic abundance and by both equilibrium and spin inversion transfer (SIT) nuclear magnetic resonance (NMR) on samples labeled at the leucine-18 alpha-carbon with 99% 13C. The data cover a wide range of temperature and concentration, and show that Jun-lz unfolds below room temperature, being far less stable than some other leucine zippers such as GCN4. 13C-NMR shows two well-separated resonances. We ascribe the upfield one to 13C spins on unfolded single chains and the downfield one to 13C spins on coiled-coil dimers. Their relative intensities provide a measure of the unfolding equilibrium constant. In SIT NMR, the recovery of the equilibrium magnetization after one resonance is inverted is modulated in part by the unfolding and folding rate constants, which are accessible from the data. Global Bayesian analysis of the equilibrium and SIT NMR data provide values for the standard enthalpy, entropy, and heat capacity of unfolding, and show the latter to be unusually large. The CD results are compatible with the NMR findings. Global Bayesian analysis of the SIT NMR data yields the corresponding activation parameters for unfolding and folding. The results show that both reaction directions are activated processes. Activation for unfolding is entropy driven, enthalpy opposed. Activation for folding is strongly enthalpy opposed and somewhat entropy opposed, falsifying the idea that the barrier for folding is solely due to a purely entropic search for properly registered partners. The activation heat capacity is much larger for folding, so almost the entire overall change is due to the folding direction. This latter finding, if it applies to GCN4 leucine zippers, clears up an extant apparent disagreement between folding rate constants for GCN4 as determined by chevron analysis and NMR in differing temperature regimes.     

A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (DeltaG(unf) = DeltaH - TDeltaS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for alpha-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30 degrees C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.     

Thermodynamic Parameters of Frog Brain κ-Opioid Receptors

Abstract: The thermodynamic parameters for [³H]-ethylketocyclazocine binding in frog ( Rana esculenta ) brain membranes have been examined. Computer-based nonlinear regression analysis of the untransformed equilibrium displacement data showed that this ligand bound to two sites with different affinities and capacities in this tissue. K _A values derived from equilibrium displacement curves have been used for calculating the changes in the standard Gibbs energy, enthalpy, and entropy during the binding process. Van't Hoff plots are bipartite, with transitions occurring at 18°C for both the high- and the low-affinity sites. For the high-affinity site, the reaction appears to be associated with a decrease in enthalpy below the transition temperature and a significant gain in entropy above this temperature. The reverse appears to be true for the low-affinity site. We conclude that this profile fairly approximates the mixed agonist-antagonist nature of this ligand and surmise that thermodynamic analysis could be a very useful tool for characterization of the nature of cloned opioid receptors in vitro.     

Thermodynamics of amino acid side-chain internal rotations

The absolute Gibbs energy, enthalpy and entropy of each of the internal rotations found in protein side chains has been calculated. The calculation requires the moments of inertia of the side chains about each bond, the potential energy barrier and the symmetry number and gives the maximum possible thermodynamic consequences of restricting side chain motion when a protein folds. Hindering side chain internal rotations is unfavourable in terms of Gibbs energy and entropy; it is enthalpically favourable at 0 K. At room temperature, it is estimated that the adverse entropy of hindering buried side chain internal rotation is only 25% of the absolute entropy. The difference between absolute entropies in the folded and unfolded states gives the entropy change for folding. The estimated Gibbs energy change for restricting each residue correlates moderately well with the probability of that residue being found on the folded protein surface, rather than in the protein interior (where motion is restricted).     

Effects of hydrated water on protein unfolding

The conformational stability of a protein in aqueous solution is described in terms of the thermodynamic properties such as unfolding Gibbs free energy, which is the difference in the free energy (Gibbs function) between the native and random conformations in solution. The properties are composed of two contributions, one from enthalpy due to intramolecular interactions among constituent atoms and chain entropy of the backbone and side chains, and the other from the hydrated water around a protein molecule. The hydration free energy and enthalpy at a given temperature for a protein of known three-dimensional structure can be calculated from the accessible surface areas of constituent atoms according to a method developed recently. Since the hydration free energy and enthalpy for random conformations are computed from those for an extended conformation, the thermodynamic properties of unfolding are evaluated quantitatively. The evaluated hydration properties for proteins of known transition temperature (Tm) and unfolding enthalpy (delta Hm) show an approximately linear dependence on the number of constituent heavy atoms. Since the unfolding free energy is zero at Tm, the enthalpy originating from interatomic interactions of a polypeptide chain and the chain entropy are evaluated from an experimental value of delta Hm and computed properties due to the hydrated water around the molecule at Tm. The chain enthalpy and entropy thus estimated are largely compensated by the hydration enthalpy and entropy, respectively, making the unfolding free energy and enthalpy relatively small. The computed temperature dependences of the unfolding free energy and enthalpy for RNase A, T4 lysozyme, and myoglobin showed a good agreement with the experimental ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein interactions with urea and guanidinium chloride. A calorimetric study.

The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at various fixed temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.     

Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites

The energetic basis of GCN4-bZIP complexes with the AP-1 and ATF/CREB sites was investigated by optical methods and scanning and isothermal titration microcalorimetry. The dissociation constant of the bZIP dimer was found to be significantly higher than that of its isolated leucine zipper domain: at 20 degrees C it is 1.45microM and increases with temperature. To avoid complications from dissociation of this dimer, DNA binding experiments were carried out using an SS crosslinked version of the bZIP. The thermodynamic characteristics of the bZIP/DNA association measured at different temperatures and salt concentrations were corrected for the contribution of refolding the basic segment upon binding, determined from the scanning calorimetric experiments. Fluorescence anisotropy titration experiments showed that the association constants of the bZIP at 20 degrees C with the AP-1 and ATF/CREB binding sites do not differ much, being 1.5nM and 6.4nM, corresponding to Gibbs energies of -49kJmol(-1) and -46kJmol(-1), respectively. Almost half of the Gibbs energy is attributable to the electrostatic component, resulting from the entropic effect of counterion release upon DNA association with the bZIP and is identical for both sites. In contrast to the Gibbs energies, the enthalpies of association of the fully folded bZIP with the AP-1 and ATF/CREB sites, and correspondingly the entropies of association, are very different. bZIP binding to the AP-1 site is characterized by a substantially larger negative enthalpy and non-electrostatic entropy than to the ATF/CREB site, implying that the AP-1 complex incorporates significantly more water molecules than the ATF/CREB complex.     

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.     

Cold unfolding of β-hairpins: a molecular-level rationalization

Isolated β-hairpins in water have a temperature dependence of their conformational stability qualitatively resembling that of globular proteins, showing both cold and hot unfolding transitions. It is shown that a molecular-level rationalization of this cold unfolding can be provided extending the approach devised for globular proteins (Graziano G. Phys Chem Chem Phys 2010; 12:14245-14252). The decrease in the solvent-excluded volume upon folding, measured by the decrease in the solvent accessible surface area, produces a gain in configurational/translational entropy of water molecules that is the main stabilizing contribution of the folded conformation. This always stabilizing Gibbs energy contribution has a parabolic-like temperature dependence in water and is exactly counterbalanced at two temperatures (i.e., the cold and hot unfolding temperatures) by the always destabilizing Gibbs energy contribution due to the loss in conformational degrees of freedom of the peptide chain.     

Thermodynamic studies on the interaction between sodium n-dodecyl sulphate and histone H2B

The thermodynamic parameters for the interaction of the anionic detergent sodium n-dodecyl sulphate (SDS) with H2B at pH 3.2, 6.4 and 10 have been measured at 27 degrees C and 37 degrees C by equilibrium dialysis to determine the Gibbs energies of detergent binding. The data have been used to obtain the enthalpy of interaction from the temperature dependence of the equilibrium constants from the Van't Hoff relation. The enthalpy of interaction between H2B and SDS is endothermic at pH 3.2, 6.4 and 10. The shapes of the enthalpy curves at pH 3.2 and 10 show some small exothermic contribution which probably indicates folding of H2B. The interactions of H2B-SDS are dominated by the increase in entropy on detergent binding. The larger negative free energy, enthalpy and entropy changes at pH 6.4 are consistent with greater denaturation relative to pH 3.2 and 10.     

Thermal unfolding in a GCN4-like leucine zipper: 13C alpha NMR chemical shifts and local unfolding curves.

13C alpha chemical shifts and site-specific unfolding curves are reported for 12 sites on a 33-residue, GCN4-like leucine zipper peptide (GCN4-lzK), ranging over most of the chain and sampling most heptad positions. Data were derived from NMR spectra of nine synthetic, isosequential peptides bearing 99% 13C alpha at sites selected to avoid spectral overlap in each peptide. At each site, separate resonances appear for unfolded and folded forms, and most sites show resonances for two folded forms near room temperature. The observed chemical shifts suggest that 1) urea-unfolded GCN4-lzK chains are randomly coiled; 2) thermally unfolded chains include significant transient structure, except at the ends; 3) the coiled-coli structure in the folded chains is atypical near the C-terminus; 4) only those interior sites surrounded by canonical interchain salt bridges fail to show two folded forms. Local unfolding curves, obtained from integrated resonance intensities, show that 1) sites differ in structure content and in melting temperature, so the equilibrium population must comprise more than two molecular conformations; 2) there is significant end-fraying, even at the lowest temperatures, but thermal unfolding is not a progressive unwinding from the ends; 3) residues 9-16 are in the lowest melting region; 4) heptad position does not dictate stability; 5) significant unfolding occurs below room temperature, so the shallow, linear decline in backbone CD seen there has conformational significance. It seems that only a relatively complex array of conformational states could underlie these findings.     

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR^?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.     

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.     

Conformation of adenosine 3',5'-monophosphate in solution as studied by the NMR-desert method. II. Self-association and temperature-dependent glycosidic isomerization at pH 7.

A study has been made of the association and the temperature-dependent conformation of adenosine 3',5'-monophosphate (cyclic AMP) in a neutral aqueous (2H2O) solution by means of proton magnetic resonance chemical shift and relaxation. The concentration and temperature-dependent chemical shifts of H(1'), H(2), and H(8), have enabled us to estimate the self-association constant, Ka = 1.1 +/- 0.3 M-1 at 25 degrees C and thermodynamic parameters delta H = -5.8 +/- 1.5 kcal/mol and delta S (25 degrees C) = -19.0 +/- 3 cal/mol per degree. The NMR-DESERT (Deuterium Substitution Effect on Relaxation Times) method has been utilized for the determination of the syn-anti conformational equilibrium in the monomeric state and for the determination of the mutual orientation of the two adenine rings in the dimeric state of cyclic AMP. The molecules were found to coexist with nearly equimolarity or syn-anti conformers and thermal activation of the molecules perturbs the syn-anti conformational equilibrium to comprise the syn form in preference at higher temperature. The glycosidic isomerization (from anti to syn) was found to be characterized both by a positive enthalpy change and by a positive entropy change. The cyclic AMP molecules prefer to take a 'trans-stacking' conformation in the dimeric state where the two molecules are arranged in such a way that the H(2) of one molecule is close to the H(8) of the other.     

Membrane protein conformational change dependent on the hydrophobic environment

Two conformational states of the coat protein of the filamentous bacteriophage M13 have been detected in detergent solution by using magnetic resonance techniques. When 3-fluorotyrosine is incorporated in place of the two tyrosine residues in the protein, four 19F nuclear magnetic resonance signals are observed, two for each conformer of the protein. The equilibrium between the two forms can be modulated by pH, temperature, and detergent structure. The rate of interconversion of the isomers is rapid on the minutes time scale but is slow relative to the T1 relaxation time of the fluorine resonances of approximately 50 ms. The conformational change between the conformers results in the perturbation of a basic residue in the protein such that this group has a pKa of approximately 9.5 in one state which shifts to 10.5 or more in the other conformational state. The temperature dependence of the equilibrium suggests an enthalpy difference of about 10 kcal/mol which is offset by entropy to give nearly zero free energy difference between the states at pH 8.3 in deoxycholate solution at room temperature. This suggests a substantial reorganization of the noncovalent interactions defining the two conformational states. The conformational equilibrium is strongly dependent on detergent structure and the presence of phospholipid in the detergent micelle. The results are not consistent with a strong, specific lipid binding to the protein but appear to be consistent with a more general effect of the overall micelle structure on the conformational state of the protein.     

Thermodynamic investigations of proteins. III. Thermodynamic description of lysozyme.

Standard functions of enthalpy, entropy and the Gibbs energy of native and denatured lysozyme in the range of 0-100 degrees C and pH 1.5-7.0 are represented in three-dimensional projections. The denaturational Gibbs energy change reaches 16 kcal mol-1 at conditions of maximal protein stability (0 degrees C, pH 4.5-7.0) and equals 14.5 kcal mol-1 at 25 degrees C and neutral pH. This result was found to be in agreement with the data reported from guanidine hydrochloride denaturation studies. Partial thermodynamic functions of the conformational and ionizational changes of the protein are obtained from entropy and Gibbs-energy changes in denaturation. The conformational partial entropy and Gibbs-energy change are found to be independent of pH. The pH-dependent partial ionizational entropy and Gibbs-energy changes are induced by normalization of the ionization behaviour of buried groups and cause a decrease of protein stability.     

Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).     

AMP and IMP binding to glycogen phosphorylase b. A calorimetric and equilibrium dialysis study

Reaction microcalorimetry and equilibrium dialysis have been used to study the binding of AMP and IMP to glycogen phosphorylase b (EC 2.4.1.1) at 25 degrees C and pH 6.9. The combination of both techniques has enabled us to obtain some of the thermodynamic parameters for these binding processes. Four binding sites were found to be present in the dimeric active enzyme for both AMP and IMP. The binding to two high-affinity sites, which, in our opinion, correspond to the activator sites, seems to be cooperative. The two low-affinity sites, which would then correspond to the inhibitor sites, appear to be independent when the nucleotides bind to the enzyme. The negative delta G0 of binding/site at 25 degrees C is the result in all cases of a balance between negative enthalpy and entropy changes. The large differences in delta H and delta S0 for the binding of AMP to the activator sites (-27 and -70 kJ mol-1; -22 and -150 J X K-1 mol-1) suggest the existence of rather extensive conformational changes taking place in phosphorylase b on binding with the allosteric activator. Whereas the affinity of AMP for the activator sites is about 1 order of magnitude higher than that of IMP, the affinity of both nucleotides, including their delta H and delta S0 values, seems to be the same for the inhibitor sites.