W0322

Intermolecular Interactions and the Thermodynamics and Kinetics of Phase Transitions in Protein Solutions. Peter G. Vekilov, Dimiter N. Petsev, Simon Brandon, Panagiotis Katsonis, Dept. of Chemical Engineering, Univ. of Houston, Houston, Texas 77204

To examine how the intermolecular interactions determine the thermodynamics and kinetics of the phase behavior in protein solutions, we investigated solutions of ferritin and apoferritinóa unique pair of proteins with identical shells but different molecular masses. Crystallization of these proteins requires the presence of two ions: Na⁺ and a divalent ion, most often Cd²⁺. Na⁺ are counterions, while Cd²⁺ form strong coordination type bonds between adjacent molecules in the lattice.

To characterize the protein-protein interactions in the presence of Na⁺ and Cd2⁺, we employed static and dynamic light scattering. The results indicate that Na⁺ ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of Cd²⁺ modifies the interaction potential - it has a repulsive part due to the Na⁺ assisted hydration sphere build-up at separations between 0.5 and 3 nm, and, after a maximum, an short range (<0.2 - 0.1 nm) attractive part due to the Cd²⁺ mediated bonds.

The specificity of the phase behavior of the two proteins includes a lack of liquid-liquid separation at temperatures between -5 to 40° C and independence of the solubility on temperature. To link the found structured potential to the phase diagram, we carried out Monte Carlo simulations. We applied Gibbs-Duhem and Gibbs Ensemble simulation techniques. The inter-molecular potential we consider is based on the a-potential (a modified, short range Lennard-Jones type potential). We additionally modified it by introducing a local maximum at separations longer than the minimum. Increasing the height of the maximum resulted in steeper liquidus lines, and eventually, in temperature-independent solubility. Another consequence of the increasing maxima was the shift of the liquid-liquid separation line to lower temperatures.

Crystallization experiments with ferritin and apoferritin revealed that the kinetic coefficient for crystallization is identical (accuracy ≤ 7%) for the pair, indicating Brownian kinetics of crystallization, i.e., the rate is determined not by the decay of an unstable intermediate, but by the rate of Brownian migration of a molecule toward a growth site. The energy barrier for attachment to a growth site is determined by the need to push aside the water molecules coating to the incoming protein molecule and the growth site. Not surprisingly, the characteristic length scale of this barrier coincides with the characteristic thickness of the water layer evidenced in the light scattering experiments.