Thermodynamics of globular proteins

The analysis of temperature-induced unfolding of proteins in aqueous solutions was performed. Based on the data of thermodynamic parameters of protein unfolding and using the method of semi-empirical calculations of hydration parameters at reference temperature 298 K, we obtained numerical values of enthalpy, free energy, and entropy which characterize the unfolding of proteins in the ‘gas phase’. It was shown that specific values of the energy of weak intramolecular bonds (?H^int), conformational free energy (?G^conf) and entropy (?S^conf) are the same for proteins with molecular weight 7–25 kDa. Using the energy value (?H^int) and the proposed approach for estimation of the conformational entropy of native protein (S^NC), numerical values of the absolute free energy (G^NC) were obtained.     

Thermodynamics of interaction of caffeic acid and quinic acid with multisubunit proteins

Helianthinin is a multisubunit protein from Sunflower seeds. Caffeic acid (CA) and quinic acid (QA) are intrinsic ligands present in sunflower seeds. The mechanism of interaction of these ligands with multisubunit proteins is limited. The present study enables one to understand the mechanism of the interaction of these ligands with the protein helianthinin. From this study, it is shown that CA has two classes of binding sites on helianthinin. The high-affinity class of sites total six from 60+/-10 for both high-affinity and low-affinity sites. Tryptophan, tyrosine and lysine residues of the protein are mainly involved in the interaction with CA. The temperature dependence of the binding in the range 10-45 degrees C can be clearly described by an enthalpy-entropy compensation effect at the low-affinity class of sites, while it is described by positive DeltaC(p)(o) at the high-affinity class of sites. This positive DeltaC(p)(o) has a contribution to the protein stability. The binding strength of CA also has a positive cooperativity at higher protein concentration. QA has two classes of binding sites on the protein based on the strength of the interaction. The interaction of QA with the protein is predominantly described by positive DeltaC(p)(o) for both classes of affinity. This suggests predominance of ionic/hydrogen bonding in the interaction process. Differential scanning calorimetric measurements reveal that the binding of both CA and QA induces destabilisation of the subunit-subunit interaction. Human methaemoglobin (mHb) has two binding sites on the molecule for CA. Both CA and QA decrease the stability of mHb, as indicated by decreased T(m). This destabilisation is also accompanied by dissociation to the monomers with concomitant conformational changes.     

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.     

Dynamics of amino acid residues in globular proteins

The dynamic differential equation model developed and tested for bovine pancreatic trypsin inhibitor and tuna ferrocytochrome c in Ponnuswamy, P.K. & Bhaskaran, R. (Int. J. Peptide Protein Res. 24, 168-179, 1984) is extended for 17 more protein crystals in this work. Average displacements are computed for 20 amino acid residues observed in 19 proteins. Detailed information on the dynamic behaviour of the individual proteins and individual residues is presented. The effect of atomic packing on the fluctuations of the amino acid residues in alpha-chymotrypsin is illustrated. A number of general points on the dynamic characteristics of globular protein molecules are presented.     

Thermodynamics of the acid transition in blue copper proteins

The thermodynamic parameters of the conformational transition occurring at low pH (acid transition, AT) in blue copper proteins, involving protonation and detachment from the Cu(I) ion of one histidine ligand, have been determined electrochemically for spinach and cucumber plastocyanins, Rhus vernicifera stellacyanin, cucumber basic protein (CBP), and Paracoccus versutus amicyanin. These data were obtained from direct protein electrochemistry experiments carried out at varying pH and temperature. For all species but CBP, the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. In particular, we found that (i) the balance of bond breaking/formation favors the acid transition in plastocyanins, which show remarkably negative DeltaH degrees '(AT) values, and (ii) the transition enthalpy turns out to be much less negative (or even positive) for the two phytocyanins (stellacyanin and CBP): for these species, the transition turns out to be observable thanks to the favorable (positive) entropy change. Thus, it is apparent that the thermodynamic "driving force" for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the free energy change of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, except for amicyanin, for which the greater exothermicity of the transition can be ascribed to peculiar features of the active site.     

A study of the preferred environment of amino acid residues in globular proteins

In the native folded conformation of a globular protein, amino acid residues distant along the polypeptide chain come together to form the compact structure. This spatial structure is such that most of the polar residues are on the surface and have contact with the solvent medium and the nonpolar residues buried in the interior which have contact with similar nonpolar side chains. This cooperativity and mutual interaction among the randomly aligned amino acid residues suggest that each type of residue may prefer to have a specific environment. To gain more insight into this aspect of residue-residue cooperativity, a detailed analysis of the preferred environment associated with each of the 20 different amino acid residues in a number of protein crystals has been carried out. The variation of nonpolar nature computed for different sizes of spheres shows that the spatial region between radii of 6 and 8 Å is more favored for hydrophobic interactions and indicates that the influence of each residue over the surrounding medium extends predominantly up to a distance of 8 Å. The analysis of the surrounding amino acid residues associated with each type of residue shows that there is a definite tendency for each type of residue to have association with specific residues. The variation in environment is found even within the polar group as well as in the nonpolar group of residues. The surrounding residues associated with isoleucine, leucine, and valine are purely nonpolar. Proline, a nonpolar residue, is often surrounded by polar residues. The surrounding nonpolar nature of the tryptophan and tyrosine residues implies that even a single polar atom in a nonpolar side chain is sufficient to reduce their hydrophobic environment. There exists a high degree of mutual residue-residue cooperativity between the pairs glutamic acid-lysine, methionine-arginine, asparagine-tryptophan, and glutamine-proline, and the mutual residue-residue noncooperativity is high for the pairs methionine-aspartic acid, cysteine-glutamic acid, histidine-glutamine, and leucine-asparagine. The formation of secondary and tertiary structures is discussed in terms of the preferred environment and mutual cooperativity among various types of amino acid residues.     

Interaction of adjacent amino acid residues in the structured regions of globular proteins

Distribution of the pairs of amino acids i, i + 1 in alpha-helical, beta-sheet and random coil regions from 46 globular proteins comprising 8115 amino acid residues was analyzed. Statistical analysis of the data excludes null hypothesis about random pairing of the amino acid residues i, i + 1 in beta-sheet and random coil configurations. The distribution of the amino acid pairs, i, i + 1 in alpha-helical configurations does not differ from the random pairing.     

The three states of globular proteins: acid denaturation.

We describe statistical mechanical theory that aims to predict protein stabilities as a function of temperature, pH, and salt concentration, from the physical properties of the constituent amino acids: (1) the number of nonpolar groups, (2) the chain length, (3) the temperature-dependent free energy of transfer, (4) the pKa's (including those in the native state) and their temperature dependencies. We calculate here the phase diagrams for apomyoglobin and hypothetical variant proteins. The theory captures essential features of protein stability including myoglobin's Tm vs pH as measured by P. L. Privalov [(1979) Advances in Protein Chemistry, Vol. 33, pp. 167-241] and its ionic strength vs pH phase diagram as measured by Y. Goto and A. L. Fink [(1990) Journal of Molecular Biology, Vol. 214, pp. 803-805]. The main predictions here are the following: (1) There are three stable states, corresponding to native (N), compact denatured (C), and highly unfolded (U), with transitions between them. (2) In agreement with experiments, the compact denatured state is predicted to have enthalpy closer to U than N because even though there is considerable hydrophobic "clustering" in C, this nevertheless represents a major loss of hydrophobic contacts relative to configurations (N) that have a hydrophobic "core." (3) C becomes more prominent in the phase diagram with increasing nonpolar content or decreasing chain length, perhaps thus accounting for (a) why lysozyme and alpha-lactalbumin differ in their denatured states, and (b) why shortened Staph nuclease molecules are compact. (4) Of major importance for protein calorimetry is Privalov's observation that the enthalpy of folding, delta H (T, pH) is independent of pH. The theory accounts for this through the prediction that the main electrostatic contribution to stability is not enthalpic; the main contribution is the entropy, mainly due to the different distributions of protons and small ions in the native and denatured states.     

Topology of globular proteins

This paper inquires whether it is reasonable to expect the native structure of proteins to be “knotted”. To this end, some topological properties of polypeptides containing disulfide bridges are discussed using notions from mathematical knot theory and graph theory. The probability of occurrence of knots in random cyclic polymers is calculated as a function of chain length by elementary Monte Carlo methods. The implications of this for protein renaturation and for determining the tertiary structure of proteins are discussed.     

Amino acid interaction preferences in proteins

Understanding the key factors that influence the interaction preferences of amino acids in the folding of proteins have remained a challenge. Here we present a knowledge‐based approach for determining the effective interactions between amino acids based on amino acid type, their secondary structure, and the contact based environment that they find themselves in the native state structure as measured by their number of neighbors. We find that the optimal information is approximately encoded in a 60 × 60 matrix describing the 20 types of amino acids in three distinct secondary structures (helix, beta strand, and loop). We carry out a clustering scheme to understand the similarity between these interactions and to elucidate a nonredundant set. We demonstrate that the inferred energy parameters can be used for assessing the fit of a given sequence into a putative native state structure.     

The binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin.

Binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin are reported. The critical micelle concentration (CMC) and the limiting solubility of 5-doxyl stearic acid were determined using the electron spin resonance (ESR)-spin label method. The CMC and the limiting solubility of this spin-label stearic acid in saline-phosphate buffer are 3.5 x 10(-5) M and 2 x 10(-4) M, respectively. We found no ESR line width evidence for pre-association of the spin-label stearate below the CMC. Maximum binding of the spin-label stearate to both bovine and human albumin occurs before micelle formation. The binding isotherm for spin-label stearic acid interaction with bovine albumin is in agreement with data obtained by others using [1-(14)C]stearic acid. For human albumin, comparison is difficult since previous data obtained with [1-(14)C]stearic acid vary widely. Comparison of the ESR 2T(||) values (the splitting between low and high field extremes, a measure of the degree of immobilization of protein-bound spin-label stearate) for bovine and human albumin indicates a greater immobilization of the spin-label molecules bound to human albumin. The binding data indicate that complexes are formed with bound spin-label stearate/albumin ratios of at least 18. The computed equilibrium constants for both bovine and human albumin indicate that the first seven spin-label molecules are tightly bound, log K > 5.0. The species predicted to form in solution by these equilibrium constants are reported.     

Moments of the distribution of the amino acid residues in tertiary structures of globular proteins

Moments of the distributions of the C^α and “side-chain atoms” and associated properties were examined in 22 globular proteins, considered as statistical aggregates of atoms. Although the distributions are generally anisotropic, the densities of the evaluated distributions are highly uniform in the interior of a single protein, as well as among the proteins investigated. The tertiary structure of proteins is characterized by a compact and uniform distribution of amino acids, independent of their molecular weight and hydrophobic character, and by an isotropic distribution of the virtual bond directions in the polypeptide folding. While the general uniformity of the density of distributions in the bulk of proteins can be justified by the architectural requirements of high thermodynamic stability, significant differences in the distribution of the C^α with respect to the “side-chain atoms” suggest a plausible explanation of the general anisotropic morphology of the proteins. The invariance of the density of distributions allows easy recognition of proteinlike domains in more complex proteins and suggests a practical way to predict the following path in single proteins.     

Disulphide bridges in globular proteins

Disulphide bridges in proteins of known sequence, connectivity and structure were studied to search for common features. Their distribution, topology, conformation and conservation were analysed in detail. Several general patterns emerge which to some extent dictate disulphide bridge formation. For example, there is a strong preference for shorter connections, with half-cystines separated by less than 24 residues in 49% of all disulphides. Right- and left-handed disulphides occur equally; the left-handed structures adopt one predominant conformation (symmetric χ₁ = ?60 °, χ₂ = ?80 °, χ₃ = t-90 °). Cystines are generally very well conserved, in contrast to cysteines, with a free —SH group, which mutate rapidly. If a disulphide is not conserved, both cystines are mutated. The role of disulphide bridges in globular proteins is discussed.     

Prediction of the structural class of globular proteins from their amino acid sequence

Two-dimensional representation of consequence of 32 proteins with known three-dimensional structure has been obtained on 20 X 20 matrix of the distribution of amino acid pairs (nearest neighbours). Prediction algorithm of the structural class of globular proteins has been worked out on the basis of the comparison of 20 X 20 matrix of the distribution of amino acid pairs for the proteins of different structural classes. The accuracy of structural class predictions of 32 proteins has been carried out (all the proteins are taken from numerous ones used to obtain the algorithm).