Peter L. Privalov

Stability of Protein Structure and Hydrophobic Interaction

Publisher Summary This chapter focuses on the stability of protein structure and hydrophobic interaction. The interest in hydrophobic interactions was stimulated by their unusual thermodynamic properties: it is believed that they are governed, not by enthalpic, but by entropic features, characterized by the undesirable entropy decrease of water in the vicinity of nonpolar groups. The amount of polar groups in proteins is almost the same as the amount of nonpolar ones; and according to crystallographic studies, most of them are arranged at distances suggesting hydrogen bond formation. Hydrogen bonds were invoked to various degrees of importance in explaining the stabilization of the native structure. The chapter examines the main achievements of microcalorimetric studies of protein denaturation and of the dissolution of nonpolar substances in water. The chapter also discusses calorimetric studies of protein denaturation. The denaturational increment of the heat capacity can be partly explained by a gradual melting of the residual structure in the denatured protein on heating. The large negative entropy of the transfer of a nonpolar substance to water at room temperature indicates a definite increase of the order in water in the presence of such solutes. Of the two approaches to decomposing the thermodynamics for dissolution of nonpolar solutes into water, the first, from a reference point at the maximum of the free energy of transfer, leads to the concept of the compact state of the nonpolar substance.

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.

Scanning microcalorimeters for studying macromolecules

Principles of scanning microcalorimeter design, their basic characteristics and microcalorimetric data processing in studying biological macromolecules in solution are discussed.

Thermodynamic study of domain organization in troponin C and calmodulin

Intramolecular melting of troponin C, calmodulin and their proteolytic fragments has been studied microcalorimetrically at various concentrations of monovalent and divalent ions. It is shown by thermodynamic analysis of the experimentally determined excess heat capacity function that the four calcium-binding domains in these two related proteins are not integrated into a single co-operative system, as would be the case if they formed a common hydrophobic core in the molecule, but still interact with each other in a very specific way. There is a positive interaction between domains I and II, which is so strong that they actually form a single co-operative block. The interaction between domains III and IV is positive also, although much less pronounced, while the interaction between the pairs of domains (I and II) and (III and IV) is negative, as if they repel each other. The structure of the co-operative block of domains I and II at room temperature does not depend noticeably on the ionic conditions, which influence its stability to a small extent only. The same applies to domain IV of calmodulin, but in troponin C this domain is unstable in the absence of divalent ions, in solutions of low ionic strength. In both proteins, the least stable is domain III, which forms a compact ordered structure at room temperature only in the presence of Ca2+. In troponin C, calcium ions can be replaced by magnesium ions, although the compact structure of domain III formed by these two ions does not seem to be quite identical. Thus, at conditions close to physiological, with regard to temperature and ionic strength, the removal of free Ca2+ from the solution induces in both proteins a reversible transition of domain III to the non-compact disordered state. This dramatic Ca2+-induced change in the domain III conformation in troponin C and calmodulin might play a key role in the functioning of these proteins as a Ca2+-controlled switch in the molecular mechanisms of living systems.

Interpreting protein/DNA interactions: distinguishing specific from non-specific and electrostatic from non-electrostatic components

We discuss the effectiveness of existing methods for understanding the forces driving the formation of specific protein–DNA complexes. Theoretical approaches using the Poisson–Boltzmann (PB) equation to analyse interactions between these highly charged macromolecules to form known structures are contrasted with an empirical approach that analyses the effects of salt on the stability of these complexes and assumes that release of counter-ions associated with the free DNA plays the dominant role in their formation. According to this counter-ion condensation (CC) concept, the salt-dependent part of the Gibbs energy of binding, which is defined as the electrostatic component, is fully entropic and its dependence on the salt concentration represents the number of ionic contacts present in the complex. It is shown that although this electrostatic component provides the majority of the Gibbs energy of complex formation and does not depend on the DNA sequence, the salt-independent part of the Gibbs energy—usually regarded as non-electrostatic—is sequence specific. The CC approach thus has considerable practical value for studying protein/DNA complexes, while practical applications of PB analysis have yet to demonstrate their merit.

Domains in human plasminogen

Calorimetric studies of intramolecular melting of human plasminogen and of its fragments under various solvent conditions show that the intact plasminogen molecule consists of seven compact co-operative subunits, which can be regarded as structural domains. Five of these domains are formed by the homologous regions, the kringles, two domains are formed by the C-terminal part of the polypeptide chain that is split at activation, forming the light chain in plasmin, while the initial 76 amino acid residue peptide does not form any compact co-operative structure. The specific influence of epsilon-aminocaproic acid on the stability of the first, the fourth and, to a lesser extent, on the second kringle domain, provides evidence that these three domains in plasminogen possess lysine-binding ability. The first four kringle domains are almost independent in the molecule, while the fifth interacts with that part of the light chain not included in either of the two domains of this chain. These two domains are of different size and co-operate strongly in plasminogen, but at its activation into plasmin they decooperate and the stability of the smaller domain, which is formed by the N-terminal part of the light chain, decreases significantly. Since the light chain is responsible for the proteolytic activity of plasmin, it becomes clear that the active site of this protein is composed of two domains, as is the case for other serine proteases.

Co-operative blocks in tropomyosin

Abstract The intramolecular melting of the α-tropomyosin coiled-coil and four of its fragments were studied by scanning microcalorimetry. It has been shown that: 1. (1) Melting of the native structure of α-tropomyosin is a complex process consisting of several discrete stages. The last stage in this process is a bimolecular reaction in the absence of the interchain S-S crosslink. 2. (2) The observed stages correspond to disruption of distinct co-operative blocks in this molecule. There are seven co-operative blocks of various sizes that can be localized unambiguously along the molecule. 3. (3) The co-operative blocks have different stabilities, which depend largely on the state of the neighbouring regions. Neighbouring blocks destabilize each other. 4. (4) The blocks from the middle part of the molecule are unstable in the absence of the interchain S-S crosslink. The conformation of this part of the tropomyosin molecule is determined by the state of the Cys190 residue. 5. (5) The co-operative regular structure does not include the terminal parts of the molecule The physical basis and biological significance of the subdivision of the tropomyosin coiled-coil into discrete co-operative blocks are discussed.

Hydration effects in protein unfolding

The enthalpies and entropies of hydration of polar, aromatic and aliphatic groups upon unfolding of nine different globular proteins were calculated over a broad temperature range using information on the three-dimensional structures of the native states of these proteins and thermodynamic data on the transfer of various low molecular compounds modeling protein groups from the gaseous phase to water. Exclusion of these hydration effects from the calorimetrically determined enthalpy and entropy of unfolding of these proteins permitted us to estimate the energy of interactions between groups packed in the interior of the native protein, and also the entropy effects associated with the increase of configurational freedom of the backbone polypeptide chain and side chains. It is shown that the compact native state of a protein is stabilized by the enthalpic interactions between internal groups while the hydration effects of all the groups, except the aliphatic ones, which are exposed upon unfolding destabilize this state.

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.

Unfolding of a Leucine zipper is not a Simple Two-state Transition

Temperature-induced unfolding of the leucine zipper, an alpha-helical, double-stranded, coiled-coil, was studied by circular dichroism spectroscopy, spectrofluorimetry and heat capacity scanning calorimetry. It is shown that this process does not represent a simple two-state transition, as previously believed, but consists of several stages. The first transition starts at the very beginning of heating from 0 degrees C and proceeds with significant heat absorption and decrease of ellipticity. This transition does not depend on the concentration of protein and is sensitive to modification of the N terminus; it is therefore associated with unfolding or fraying of this part of the leucine zipper. The second transition takes place at a considerably higher temperature; it is more pronounced than the first one and does not depend on the concentration of protein, i.e. it is unimolecular. This transition is sensitive to modification of both termini of the leucine zipper and affects the optical properties of a tryptophan residue placed in the central part of the zipper. It therefore involves the whole dimer but does not result in its dissociation, presumably being associated with some repacking of the coiled-coil. This second transition is followed at higher temperatures by the concentration-dependent cooperative unfolding/dissociation of the two strands. The enthalpy and entropy of the temperature-induced structural changes of the leucine zipper that take place before its cooperative unfolding/dissociation comprises almost 40% of the total enthalpy and entropy of unfolding of the completely folded coiled-coil, the state in which it appears to be below 0 degrees C. Comparison of the total enthalpy of leucine zipper unfolding with that of a single-stranded alpha-helix shows that their temperature-dependence correlates with the extent of intramolecular non-polar contacts and allows an assessment of the enthalpy of hydrogen bonding in alpha-helices, which appears to be about 3.3kJmol(-1) at 20 degrees C.