The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase.

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.     

Site-directed mutagenesis of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase.

Site-specific mutants have been produced in order to investigate the role of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the 'hinge' bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild-type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-distant interactions are more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild-type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanism of the enzyme.     

Comparison of proteolytic susceptibility in phosphoglycerate kinases from yeast and E. coli: modulation of conformational ensembles without altering structure or stability

Escherichia coli phosphoglycerate kinase (PGK) is resistant to proteolytic cleavage while the yeast homolog from Saccharomyces cerevisiae is not. We have explored the biophysical basis of this surprising difference. The sequences of these homologs are 39% identical and 56% similar. Determination of the crystal structure for the E. coli protein and comparison to the previously solved yeast structure reveals that the two proteins have extremely similar tertiary structures, and their global stabilities determined by equilibrium denaturation are also very similar. The extrapolated unfolding rate of E. coli PGK is, however, 10(5) slower than that of the yeast homolog. This surprisingly large difference in unfolding rates appears to arise from a divergence in the extent of cooperativity between the two structural domains (the N and C-domains) that make up these kinases. This is supported by: (1) the C-domain of E. coli PGK cannot be expressed or fold independently of the N-domain, while both domains of the yeast protein fold in isolation into stable structures and (2) the energetics and kinetics of the proteolytically sensitive state of E. coli PGK match those for global unfolding. This suggests that proteolysis occurs from the globally unfolded state of E. coli PGK, while the characteristics defining the yeast homolog suggest that proteolysis occurs upon unfolding of only the C-domain, with the N-domain remaining folded and consequently resistant to cleavage.     

Thermodynamic study of phosphoglycerate kinase from Thermotoga maritima and its isolated domains: reversible thermal unfolding monitored by differential scanning calorimetry and circular dichroism spectroscopy

The folding of phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima and its isolated N- and C-terminal domains (N1/2 and C1/2) was characterized by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. At pH 3.0-4.0, reversible thermal denaturation of TmPGK occurred below 90 degrees C. The corresponding peaks in the partial molar heat capacity function were fitted by a four-state model, describing three well-defined unfolding transitions. Using CD spectroscopy, these are ascribed to the disruption of the domain interactions and subsequent sequential unfolding of the two domains. The isolated N-terminal domain unfolds reversibly between pH 3.0 and pH 4.0 to >90% and at pH 7.0 to about 70%. In contrast, the isolated engineered C-terminal domain only shows reversible thermal denaturation between pH 3.0 and pH 3.5. Neither N1/2 nor C1/2 obeys the simple two-state mechanism of unfolding. Instead, both unfold via a partially structured intermediate. In the case of N1/2, the intermediate exhibits native secondary structure and perturbed tertiary structure, whereas for C1/2 the intermediate could not be defined with certainty.     

Dodine as a transparent protein denaturant for circular dichroism and infrared studies

The fungicide dodine combines the cooperative denaturation properties of guanidine with the mM denaturation activity of SDS. It was previously tested only on two small model proteins. Here we show that it can be used as a chemical denaturant for phosphoglycerate kinase (PGK), a much larger two‐domain enzyme. In addition to its properties as a chemical denaturant, dodine facilitates thermal denaturation of PGK, and we show for the first time that it also facilitates pressure denaturation of a protein. Much higher quality circular dichroism and amide I′ infrared spectra of PGK can be obtained in dodine than in guanidine, opening the possibility for use of dodine as a denaturant when UV or IR detection is desirable. One caution is that dodine denaturation, like other detergent‐based denaturants, is less reversible than guanidine denaturation.     

Crystal structures of putative phosphoglycerate kinases from B. anthracis and C. jejuni

Phosphoglycerate kinase (PGK) is indispensable during glycolysis for anaerobic glucose degradation and energy generation. Here we present comprehensive structure analysis of two putative PGKs from Bacillus anthracis str. Sterne and Campylobacter jejuni in the context of their structural homologs. They are the first PGKs from pathogenic bacteria reported in the Protein Data Bank. The crystal structure of PGK from Bacillus anthracis str. Sterne (BaPGK) has been determined at 1.68 ? while the structure of PGK from Campylobacter jejuni (CjPGK) has been determined at 2.14 ? resolution. The proteins' monomers are composed of two domains, each containing a Rossmann fold, hinged together by a helix which can be used to adjust the relative position between two domains. It is also shown that apo-forms of both BaPGK and CjPGK adopt open conformations as compared to the substrate and ATP bound forms of PGK from other species.     

A comparative thermodynamic study of heat and cold denaturation of beta-lactoglobulin]

The changes in structure and thermodynamic parameters of beta-lactoglobulin upon heat and cold denaturation have been studied using both scanning microcalorimetry and circular dichroism spectroscopy methods. It has been shown that in contrast to the heat denaturation process, the cold denaturation of beta-lactoglobulin is accompanied by an opposite heat effect. In all cases, the calorimetrically measured enthalpy of beta-lactoglobulin cold denaturation is higher than it was expected from the two-state model of denaturation transition. It has been concluded that beta-lactoglobulin cold denaturation cannot be represented by a transition between two microscopic states--native and denatured. The latter, is due to the additional process that occurs together with the disruption of the beta-lactoglobulin tertiary structure and is accompanied by increasing heat capacity. Taking into account the heat capacity contribution of this process upon calculation of the enthalpy makes it closer to the enthalpy value calculated for the two-state model of denaturation transition.     

A study of the hinge-bending mechanism of yeast 3-phosphoglycerate kinase.

The hinge-bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site-directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C-terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding. The effect of substrates on the conformation of PGK was studied using 8-anilino-1-naphthalenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C-terminal domain, and 8-(2-[(iodoacetyl)ethyl]amino)naphthalene (I-AEDANS), attached to Cys197 on the N-terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1-5 degrees change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS. The enzyme activity of PGK shows a break in the Arrhenius plot at 20 degrees C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine. The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.     

Kietics of thermal unfolding and refolding of thermostable phosphoglycerate kinase.

The kinetics of denaturation by guanidine hydrochloride (GuHCl) of a thermostable phosphoglycerate kinase (PGK) extracted from Thermus thermophilus and of yeast PGK at neutral pH were studied by circular dichroism. Denaturation by GuHCl proceeded as a first-order reaction. The activation free energy of the denaturation reactions (delta Gf not identical to ) in the absence of GuHCl was estimated to be 32.7 kcal/mol for T. thermophilus PGK and 27.9 kcal/mol for yeast PGK (at 25 degrees C). Measurements of the rate constants at various temperatures indicated that delta Gf not identical to has maximum values at 29 degrees C for T. thermophilus PGK and at 20 degrees C for yeast PGK, and that the temperature dependences of delta Gf not identical to, delta Hf not identical to, and delta Sf not identical to for T. thermophilus PGK are smaller than those of yeast PGK. Values of delta Sf not identical to for thermal denaturation for both PGK's are approximately 200 e.u.     

Number of cooperative regions (energy domains) in the pepsin molecule depends on the pH of the medium]

Ethanol and pH influence on the number and dimensions of cooperative regions in pepsin molecule was studied by scanning microcalorimetry. It is shown that ethanol solution causes a decrease of temperature of protein denaturation but does not influence the number of energetic domains. While changing pH from 6.7 to 2.0 the number of thermodynamic cooperative units (defined as the ratio delta Hcal/delta Heff) decreases from four to two. This process, as shown by CD technique, is followed by changes neither in the secondary structure, nor in the local environment of aromatic amino acids. A conclusion is made that the distinctions in cooperative characteristics of the protein globule at different pH are determined by electrostatic interactions of separate parts of the molecule.     

Characterization of a quaternary-structured folding intermediate of an antibody Fab-fragment.

Antibody folding is a complex process comprising folding and association reactions. Although it is usually difficult to characterize kinetic folding intermediates, in the case of the antibody Fab fragment, domain-domain interactions lead to a rate-limiting step of folding, thus accumulating folding intermediates at a late step of folding. Here, we analyzed a late folding intermediate of the Fab fragment of the monoclonal antibody MAK 33 from mouse (kappa/IgG1). As a strategy for accumulation of this intermediate we used partial denaturation of the native Fab by guanidinium chloride. This denaturation intermediate, which can be populated to about 90%, is indistinguishable from a late-folding intermediate with respect to denaturation and renaturation kinetics. The spectroscopic analysis reveals a native-like secondary structure of this intermediate with aromatic side chains only slightly more solvent exposed than in the native state. The respective partner domains are weekly associated. From these data we conclude that the intramolecular association of the two chains during folding, with all domains in a native-like structure, follows a two-step mechanism. In this mechanism, presumably hydrophobic interactions are followed by rearrangements leading to the exact complementarity of the contact sites of the respective domains.     

Structural energetics of barstar studied by differential scanning microcalorimetry.

The energetics of barstar denaturation have been studied by CD and scanning microcalorimetry in an extended range of pH and salt concentration. It was shown that, upon increasing temperature, barstar undergoes a transition to the denatured state that is well approximated by a two-state transition in solutions of high ionic strength. This transition is accompanied by significant heat absorption and an increase in heat capacity. The denaturational heat capacity increment at approximately 75 degrees C was found to be 5.6 +/- 0.3 kJ K-1 mol-1. In all cases, the value of the measured enthalpy of denaturation was notably lower than those observed for other small globular proteins. In order to explain this observation, the relative contributions of hydration and the disruption of internal interactions to the total enthalpy and entropy of unfolding were calculated. The enthalpy and entropy of hydration were found to be in good agreement with those calculated for other proteins, but the enthalpy and entropy of breaking internal interactions were found to be among the lowest for all globular proteins that have been studied. Additionally, the partial specific heat capacity of barstar in the native state was found to be 0.37 +/- 0.03 cal K-1 g-1, which is higher than what is observed for most globular proteins and suggests significant flexibility in the native state. It is known from structural data that barstar undergoes a conformational change upon binding to its natural substrate barnase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric investigations of the structural stability and interactions of colicin B domains in aqueous solution and in the presence of phospholipid bilayers

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the full-length toxin is consistent with two endothermic transitions. Whereas peak A (T(m) = 55 degrees C) most likely corresponds to the receptor/translocation domain, peak B (T(m) = 59 degrees C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 degrees C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T(m) = 65 degrees C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B ( approximately 40%) or its pore-forming fragment ( approximately 80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T(m) = 54 degrees C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.     

Evaluation of riboflavin binding protein domain interaction using differential scanning calorimetry

Riboflavin binding (or carrier) protein (RfBP) is a monomeric, two-domain protein, originally purified from hens' egg white. RfBP contains nine disulfide bridges; as a result, the protein forms a compact structure and undergoes reversible three-state thermal denaturation. This was demonstrated using a differential scanning calorimetry (DSC) method [Wasylewski M. (2000) J. Prot. Chem. 19(6), 523-528]. It has been shown that the RfBP complex with riboflavin denaturates in a three-state process which may be attributed to sequential unfolding of the RfBP domains. In case of apo RfBP, the ligand binding domain denaturates at a lower temperature than the C-terminal domain. Ligand binding greatly enhances the thermostability of the N-terminal domain, whereas the C-terminal domain thermostability is only slightly affected and, in case of the examined holo RfBPs, the denaturation peaks of both domains merge or cross over. The magnitude of the changes depends on ligand structure. A detailed study of protein concentration effects carried out in this work allowed to estimate not only the thermostability of both domains but also the strength of domain interactions. The DeltaCp, of denaturation was found for C-terminus and N-terminus of RfBP-riboflavin complex to amount to 2.5 and -1.9 kcal mol(-1), respectively. The calculated domain interaction free energy, DeltaGCN, was estimated to be approximately -1580 cal mol(-1) at 67.0 degrees C. This value indicates that the interdomain interaction is of medium strength.     

Co-evolution of proteins with their interaction partners

The divergent evolution of proteins in cellular signaling pathways requires ligands and their receptors to co-evolve, creating new pathways when a new receptor is activated by a new ligand. However, information about the evolution of binding specificity in ligand-receptor systems is difficult to glean from sequences alone. We have used phosphoglycerate kinase (PGK), an enzyme that forms its active site between its two domains, to develop a standard for measuring the co-evolution of interacting proteins. The N-terminal and C-terminal domains of PGK form the active site at their interface and are covalently linked. Therefore, they must have co-evolved to preserve enzyme function. By building two phylogenetic trees from multiple sequence alignments of each of the two domains of PGK, we have calculated a correlation coefficient for the two trees that quantifies the co-evolution of the two domains. The correlation coefficient for the trees of the two domains of PGK is 0. 79, which establishes an upper bound for the co-evolution of a protein domain with its binding partner. The analysis is extended to ligands and their receptors, using the chemokines as a model. We show that the correlation between the chemokine ligand and receptor trees' distances is 0.57. The chemokine family of protein ligands and their G-protein coupled receptors have co-evolved so that each subgroup of chemokine ligands has a matching subgroup of chemokine receptors. The matching subfamilies of ligands and their receptors create a framework within which the ligands of orphan chemokine receptors can be more easily determined. This approach can be applied to a variety of ligand and receptor systems.     

A pH dependence study on the unfolding and refolding of apoazurin

Azurin, a small blue copper protein from the bacterial species Pseudomonas aeruginosa, is mostly a β-sheet protein arranged into a single domain. Previous folding studies have shown that the equilibrium denaturation of the holoprotein follows a two-state process; however, upon removal of the copper, the denaturation had been reported to follow a three-state process. The two unfolding transitions measured for apoazurin had been thought to arise from two different folding domains. However, in the present work, we found that the denaturation of apoazurin occurs over a single transition and we determined the folding free energy to be −27.8±2.4 kJ mol⁻¹. From this measurement along with measurements previously reported for the unfolding of the holoazurin, we were able to determine that Cu(II) and Cu(I) stabilize the native structure by 25.1±6.9 kJ/mol and 12.9±8.1 kJ/mol, respectively. It is our contention that the second transition displayed in the denaturation curves previously reported for apoazurin arise from protein heterogeneity—in particular, from the presence of Zn(II) azurin. We extended our investigation into the denaturation of Zn(II) azurin at pH 6.0 and 7.5. The equilibrium denaturation studies show that the zinc ion significantly stabilizes the native-state structure at pH 7.5 and very little at the lower pH. We attribute the decrease in the stabilizing effect of the zinc ion with decreasing pH to the protonation of two histidinyl side chains. When protonated the ligands, His 46 and His 117, are incapable of binding a metal ion. Further, comparing the denaturation curves of Zn(II) azurin measured by circular dichroism with those measured by fluorescence indicates that the denaturation of Zn(II) azurin is far less simple than the denaturation of apoazurin.     

Apo-parvalbumin as an intrinsically disordered protein

Recently defined family of intrinsically disordered proteins (IDP) includes proteins lacking rigid tertiary structure meanwhile fulfilling essential biological functions. Here we show that apo-state of pike parvalbumin (alpha- and beta-isoforms, pI 5.0 and 4.2, respectively) belongs to the family of IDP, which is in accord with theoretical predictions. Parvalbumin (PA) is a 12-kDa calcium-binding protein involved into regulation of relaxation of fast muscles. Differential scanning calorimetry measurements of metal-depleted form of PA revealed the absence of any thermally induced transitions with measurable denaturation enthalpy along with elevated specific heat capacity, implying the lack of rigid tertiary structure and exposure of hydrophobic protein groups to the solvent. Calcium removal from the PAs causes more than 10-fold increase in fluorescence intensity of hydrophobic probe bis-ANS and is accompanied by a decrease in alpha-helical content and a marked increase in mobility of aromatic residues environment, as judged by circular dichroism spectroscopy (CD). Guanidinium chloride-induced unfolding of the apo-parvalbumins monitored by CD showed the lack of fixed tertiary structure. Theoretical estimation of energetics of the charge-charge interactions in the PAs indicated their pronounced destabilization upon calcium removal, which is in line with sequence-based predictions of disordered protein chain regions. Far-UV CD studies of apo-alpha-PA revealed hallmarks of cold denaturation of the protein at temperatures below 20 degrees C. Moreover, a cooperative thermal denaturation transition with mid-temperature at 10-15 degrees C is revealed by near-UV CD for both PAs. The absence of detectable enthalpy change in this temperature region suggests continuous nature of the transition. Overall, the theoretical and experimental data obtained show that PA in apo-state is essentially disordered nevertheless demonstrates complex denaturation behavior. The native rigid tertiary structure of PA is attained upon association of one (alpha-PA) or two (beta-PA) calcium ions per protein molecule, as follows from calorimetric and calcium titration data.