D. N. Petsev

Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes.

We have studied the structure of the protein species and the protein-protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution.

Diffusion-limited kinetics of the solution–solid phase transition of molecular substances

For critical tests of whether diffusion-limited kinetics is an option for the solution–solid phase transition of molecular substances or whether they are determined exclusively by a transition state, we performed crystallization experiments with ferritin and apoferritin, a unique pair of proteins with identical shells but different molecular masses. We find that the kinetic coefficient for crystallization is identical (accuracy ≤7%) for the pair, indicating diffusion-limited kinetics of crystallization. Data on the kinetics of this phase transition in systems ranging from small-molecule ionic to protein and viri suggest that the kinetics of solution-phase transitions for broad classes of small-molecule and protein materials are diffusion-limited.

Intermolecular Interactions, Nucleation and Thermodynamics of Crystallization of Hemoglobin C

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.

Solvent entropy contribution to the free energy of protein crystallization.

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(-1) x K(-1), 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.

Temperature-independent solubility and interactions between apoferritin monomers and dimers in solution

Abstract We used chromatographic, static and dynamic light scattering techniques, and atomic force microscopy (AFM) to study the structure of the protein species and the protein–protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of NaAc buffer and CdSO 4 . The sizes and shapes of the monomers and dimers, separated by size-exclusion chromatography, were determined by dynamic light scattering and AFM. While the monomer is an apparent sphere with a diameter corresponding to previous X-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was used to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The addition of even small amounts of Cd 2+ causes attraction between the monomer molecules. Furthermore, we found that the second virial coefficient and the protein solubility do not noticeably depend on temperature in the range from 0°C to 40°C. This suggests that the enthalpy for crystallization of apoferritin is close to zero, and the gain of entropy is essentially constant in this temperature range. We also found that in solutions of the apoferritin dimer, the molecules attract even in the presence of acetate buffer only, indicating a change in the surface of the apoferritin molecule. In view of the repulsion between the monomers at the same conditions, this suggests that the dimers and higher oligomers form only after partial unfolding of some of the apoferritin subunits. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization in the protein solution.

Micellization and interfacial properties of alkyloxyethylene sulfate surfactants in the presence of multivalent counterions

Alkyloxyethylene sulfates are a special class of surfactants that are unusually stable in the presence of multivalent counterions and are not as prone to precipitation as anionic surfactants without intermediate ethoxy groups in the molecule. However, formation of micelles, their structure, and the properties of monolayers of these surfactants exhibit very interesting and sometimes unexpected properties depending on the nature of the ions dissolved in the solution. This paper presents a brief overview of our recent efforts to reveal the nature of these properties, including some new results. We show that the strong binding of multivalent (and particularly trivalent counterions) triggers a sphere-to-cylinder shape transition of the micelles and facilitates their further growth, even at very low ionic strength. The properties of surfactant monolayers are coupled to those of the micelles in the bulk and are governed also by multivalent counterion binding. The effect of multivalent counterions on the aggregation and structure formation in anionic surfactant solutions has both fundamental and practical importance.