Stanley J. Gill

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.

Solid model compounds and the thermodynamics of protein unfolding

Analysis of thermodynamic data on the dissolution of solid cyclic dipeptides into water in terms of group additivity provides a rationale for the enthalpy and entropy convergence temperatures observed for small globular protein denaturation and the dissolution of model compounds into water. Convergence temperatures are temperatures at which the extrapolated enthalpy or entropy changes for a series of related compounds take on a common value. At these temperatures (TH* and TS*) the apolar contributions to the corresponding thermodynamic values (delta H degrees and delta S degrees) are shown to be zero. Other contributions such as hydrogen bonding and configurational effects can then be evaluated and their quantitative effects on the stability of globular proteins assessed. It is shown that the denaturational heat capacity is composed of a large positive contribution from the exposure of apolar groups and a significant negative contribution from the exposure of polar groups in agreement with previous results. The large apolar contribution suggests that a liquid hydrocarbon model of the hydrophobic effect does not accurately represent the apolar contribution to delta H degrees of denaturation. Rather, significant enthalpic stabilizing contributions are found to arise from peptide groups (hydrogen bonding). Combining the average structural features of globular proteins (i.e. number of residues, fraction of buried apolar groups and fraction of hydrogen bonds) with their specific group contributions permits a first-order prediction of the thermodynamic properties of proteins. The predicted values compare well with literature values for cytochrome c, myoglobin, ribonuclease A and lysozyme. The major thermodynamic features are described by the number of peptide and apolar groups in a given protein.

Heats of solution of gaseous hydrocarbons in water at 25°C

The heats of solution at 25°C for a number of hydrocarbon gases are reported as measured by a calorimetric method. There is excellent agreement between the standard enthalpy changes of solution measured calorimetrically and those derived from high precision temperature dependent solubility measurements. However the calorimetrically determined standard enthalpies of solution of a number of gases are greatly improved over values obtained from low precision temperature dependent solubility measurements. A method is presented to readily estimate the standard errors in the standard enthalpy change for any process derived from the temperature dependence of the equilibrium constant for the process. Comparison of the standard enthalpies and entropies of solution of hydrocarbon gases in water shows that the standard free energies of solution for all hydrocarbon gases investigated are dominated by unfavorable entropy contributions. A strong linear correlation between the standard entropy of solution and the number of hydrogens in the hydrocarbon molecule is found. This correlation suggests that the hydrocarbon hydrophobic effect is regulated by the number of allowable configurations of a water molecule in contact with each C−H group.

Generalized binding phenomena in an allosteric macromolecule

A general macromolecular partition function is developed in terms of chemical ligand activity, temperature and pressure for systems described by an array of species which are characterized by their state of allosteric conformation and ligand stoichiometry. The effects of chemical ligand binding, enthalpy change, and volume change are treated in a parallel manner. From a broad viewpoint all of these effects can be regarded as specific cases of generalized binding phenomena. This approach provides a general method for analyzing calorimetric and ligand binding experiments. Several applications are given: (1) Thermal scanning data for tRNAphe (P.L. Privalov and V.V. Filimonov, J. Mol. Biol. 122 (1978) 447) are shown to fit a general model with six conformational states. By application of linkage theory it is shown that sodium chloride is expelled as the molecule denatures. (2) The results of calorimetric titrations on the arabinose binding protein (H. Fukada, J.M. Sturtevant and F.A. Quiocho, J. Mol. Biol. 258 (1983) 13193) are shown to fit a simple two-state allosteric model. (3) A thermal binding curve is simulated for an unusual respiratory protein, trout I hemoglobin (B.G. Barisas and S.J. Gill, Biophys. Chem. 9 (1979) 235), in order to illustrate both the similarities and differences between enthalpy and chemical ligand binding processes.

Heats of solution of gaseous hydrocarbons in water at 15, 25, and 35°C

Calorimetrically measured heats of solution of eleven hydrocarbon gases into water are reported at 15 and 25°C. Gases studied are methane, ethane, propane, n-butane, 2-methylpropane, 2,2-dimethylpropane, cyclopropane, ethene, propene, 1-butene, and ethyne. These values in combination with previous results are used to derive heat capacity changes at 25°C. Comparison of enthalpy and heat capacity values with those from other studies shows satisfactory agreement. Correlation of the heat capacity change with the number of water molecules in the first solvation shell of the solute suggests that the observed heat capacity changes are primarily due to changes in the water molecules in this solvent shell.

A twin titration microcalorimeter for the study of biochemical reactions

A small-volume (200 microliter) titration calorimeter of high sensitivity (1 mu cal ) has been developed for the purpose of studying biochemical reactions where the amounts of material are limited to a few nanomoles. High sensitivity is achieved by calorimetric twining , use of glass cells, elimination of vapor space, effective low-energy stirring, and reduction of measurement time. The calorimeter operates using the heat conduction principal with computer-controlled electrical compensation, which reduces the measurement time of each point from 10 to 3 min. This reduction in time is accompanied by a corresponding increase in the precision of measurement. The use of the calorimeter is demonstrated by a measurement of the heat of oxygenation of hemocyanin.

Enthalpies of aqueous solution of noble gases at 25°C

The standard enthalpies of solution of rare gases (helium, neon, argon, krypton, and xenon) in water at 25°C have been measured by a high precision steady-state calorimetric method. The aqueous solvation process is energetically favorable at 25°C for the gases studied. Values of the standard free energy, enthalpy, and entropy changes are found to be well correlated with cavity surface areas and the number of water molecules in the first solvation shell. Also, the values of the standard enthalpy and entropy of solution for the rare gases are found to have the same dependence on the number of solvation shell water molecules as inorganic and hydrocarbon gases. These results imply that the dominant source of enthalpy and entropy change resides in the first solvation shell.

The balance sheet of a hemoglobin. Thermodynamics of CO binding by hemoglobin trout I.

Direct step-by-step calorimetric measurements of the heat of combination of trout I hemoglobin with CO, together with a closely related set of measurements of the CO binding curves at 20°C and 4°C, make it possible to establish a complete thermodynamic balance sheet for the process within the framework of the concerted model. The results show that the introduction of ligand into a subunit when the molecule is in the T state is characterized by a positive value of ΔH , probably due to an endothermic tertiary conformational change which is conditioned by the presence of the subunit in the T state. The final value of ΔH , realized when the molecule is in the R state, is negative, as is the value of ΔH for the reaction of human hemoglobin with oxygen over the whole range of saturations. This change of sign as shown by trout hemoglobin represents a striking difference between the properties, and presumably the structures, of the two hemoglobins.

Oxygen binding to sickle cell hemoglobin.

Abstract The extent of oxygen binding and light scattering of concentrated solutions of hemoglobin S have been determined as a function of oxygen partial pressure using a thin film optical cell. Nearly reversible oxygen binding is observed as witnessed by the small hysteresis found between slow deoxygenation and reoxygenation runs. High co-operativity is noted from unusually large concentration-dependent Hill coefficients when aggregated hemoglobin S is present. The application of linkage theory with the inclusion of non-ideal solution properties permits a test of various simple models for oxygen binding to both the monomer ( α 2 β 2 s ) and polymer (aggregated) phase. It is concluded that oxygen binding to the polymer is either negligible or small under present experimental conditions. Phase diagrams of the solution concentration in equilibrium with polymer phase as a function of oxygen partial pressure are derived using best fit values of polymer parameters.

Thermodynamics of dissolution of solid cyclic dipeptides containing hydrophobic side groups

Abstract The effect of hydrophobic side chains on the dissolution properties of cyclic dipeptides (diketopiperazines) has been studied by titration microcalorimetry as a function of temperature in the range 288 to 313 K. The enthalpy and heat capacity per amino acid residue are roughly additive and scale linearly with the water-accessible surface area. Application of these results to the estimation of heat-capacity changes upon denaturation of globular proteins supports the assertion that the protein interior behaves as an intramolecular solid and that the denaturational heat-capacity change results from the hydration of hydrophobic groups.