We show with three proteins that trapping and release of the water molecules upon crystallization is a determinant of the crystallization thermodynamics. With HbC, a strong retrograde solubility dependence on temperature yields a high positive enthalpy of 155 kJ mol -1 , i.e., crystallization is only possible because of the huge entropy gain of 610 J mol, stemming from the release of up to 10 water molecules per protein intermolecular contact. With apoferritin, the enthalpy of crystallization is close to zero. The main component in the crystallization driving force is the entropy gain due to the release upon crystallization of two water molecules bound to one protein molecules in solution. With both proteins, the density of the growth sites imaged by AFM is in excellent agreement with a calculation using the crystallization free energy. With lysozyme, the entropy effect due to the restructuring of the water molecules is negative. This leads to higher solubility.

The mutated hemoglobin HbC (beta 6 Glu-->Lys), in the oxygenated (R) liganded state, forms crystals inside red blood cells of patients with CC and SC diseases. Static and dynamic light scattering characterization of the interactions between the R-state (CO) HbC, HbA, and HbS molecules in low-ionic-strength solutions showed that electrostatics is unimportant and that the interactions are dominated by the specific binding of solutions' ions to the proteins. Microscopic observations and determinations of the nucleation statistics showed that the crystals of HbC nucleate and grow by the attachment of native molecules from the solution and that concurrent amorphous phases, spherulites, and microfibers are not building blocks for the crystal. Using a novel miniaturized light-scintillation technique, we quantified a strong retrograde solubility dependence on temperature. Thermodynamic analyses of HbC crystallization yielded a high positive enthalpy of 155 kJ mol(-1), i.e., the specific interactions favor HbC molecules in the solute state. Then, HbC crystallization is only possible because of the huge entropy gain of 610 J mol(-1) K(-1), likely stemming from the release of up to 10 water molecules per protein intermolecular contact-hydrophobic interaction. Thus, the higher crystallization propensity of R-state HbC is attributable to increased hydrophobicity resulting from the conformational changes that accompany the HbC beta 6 mutation.

We have developed a miniaturized (volume of crystallizing solution ∼100 μl) technique for the determination of protein solubility as a function of temperature. After nucleation, crystals are detected by the light they scatter. Then the temperature at which a solution with the initial concentration is in equilibrium with the crystals is sought by stepwise, equilibrium dissolution of the crystals. The approach to solubility from the side of dissolution provides for higher accuracy of the determinations. The method was used to determine the temperature dependence of the solubility of human hemoglobin (Hb) C, for which high-resolution x-ray crystallography data are needed to understand the structural basis for the drastically different in vivo aggregation/crystallization behavior of β6 Hb mutants.