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Trimethylamine n-oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins and offsets the destabilizing effects of urea. To investigate the molecular mechanism of these effects, we have studied the thermodynamics of interaction between TMAO and protein functional groups. The solubilities of a homologous series of cyclic dipeptides were measured by differential refractive index and the dissolution heats were determined calorimetrically as a function of TMAO concentration at 25 degrees C. The transfer free energy of the amide unit (-CONH-) from water to 1 M TMAO is large and positive, indicating an unfavorable interaction between the TMAO solution and the amide unit. This unfavorable interaction is enthalpic in origin. The interaction between TMAO and apolar groups is slightly favorable. The transfer free energy of apolar groups from water to TMAO consists of favorable enthalpic and unfavorable entropic contributions. This is in contrast to the contributions for the interaction between urea and apolar groups. Molecular dynamics simulations were performed to provide a structural framework for the interpretation of these results. The simulations show enhancement of water structure by TMAO in the form of a slight increase in the number of hydrogen bonds per water molecule, stronger water hydrogen bonds, and long-range spatial ordering of the solvent. These findings suggest that TMAO stabilizes proteins via enhancement of water structure, such that interactions with the amide unit are discouraged.

Protein unfolding and the dissolution of hydrophobic compounds (including solids, liquids, and gases) in water are characterized by a linear relation between entropy change and heat capacity change. The same slope is found for various classes of compounds, whereas the intercept depends on the particular class. The feature common to these processes is exposure of hydrophobic groups to water. These observations make possible the assignment of the heat capacity change to hydrophobic solvation and lead to the description of protein stability in terms of a hydrophobic and a nonhydrophobic contribution. A general representation of protein stability is given by the heat capacity change and the temperature.

A theoretical development in the evaluation of proton linkage in protein binding reactions by isothermal titration calorimetry (ITC) is presented. For a system in which binding is linked to protonation of an ionizable group on a protein, we show that by performing experiments as a function of pH in buffers with varying ionization enthalpy, one can determine the pK(a)'s of the group responsible for the proton linkage in the free and the liganded states, the protonation enthalpy for this group in these states, as well as the intrinsic energetics for ligand binding (delta H(o), delta S(o), and delta C(p)). Determination of intrinsic energetics in this fashion allows for comparison with energetics calculated empirically from structural information. It is shown that in addition to variation of the ligand binding constant with pH, the observed binding enthalpy and heat capacity change can undergo extreme deviations from their intrinsic values, depending upon pH and buffer conditions.

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The ability to predict the strength of the association of peptide hormones or other ligands with their protein receptors is of fundamental importance in the fields of protein engineering and rational drug design. To form a tight complex between a flexible peptide hormone and its receptor, the largeloss of configurational entropy must be overcome. Recently, the crystallographic structure of the complex between angiotensin II and the Fab fragment of a high affinity monoclonal antibody has been determined (Garcia, K.C., Ronco, P.M., Verroust, P.J., Brünger, A.T., Amzel, L.M. Three-dimensional structure of an angiotensin II-Fab complex at 3 A: Hormone recognition by an anti-idiotypic antibody. Science 257:502-507, 1992). In this paper we present a study of the thermodynamics of the association by high sensitivity isothermal titration calorimetry. The results of the experiments indicate that at 30 degrees C the binding is characterized by (1) a delta H of -8.9 +/- 0.7 kcal mol-1, (2) a delta Cp of -240 +/- 20 cal K-1 mol-1, and (3) the release of 1.1 +/- 0.1 protons per binding site in the pH range 6.0-7.3. Using these values and the previously determined binding constant in phosphate buffer, delta G at 30 degrees C is estimated as -11 kcal mol-1 and delta S as 6.9 cal K-1 mol-1. The calorimetric data indicate that binding is favored both enthalpically and entropically. These results have been complemented by structural thermodynamic calculations. The calculated and experimentally determined thermodynamic quantities are in good agreement.(ABSTRACT TRUNCATED AT 250 WORDS)

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