Studies of macromolecular crowding

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1. We are attempting to improve our previously published theoretical models of the effect of macromolecular crowding upon protein associations and isomerizations. Current work focuses on the statistical-thermodynamic formulation of a potential of average force acting between two "tracer" hard particles in a fluid containing an arbitary volume fraction of "crowder" hard particles. This is not an analytically soluble problem, and we are trying a variety of approximate techniques in order to arrive at a robust solution that is not critically dependent upon the nature of the approximations made in the formulation of the model. We have carried out calculations and compared the results of the theoretical models with the results of Monte Carlo and/or molecular dynamics simulation (by N. Dokholyan). The results indicate agreement between theory and simulation at contact distance between the tracer particles, but discrepancies at larger interparticle distances that are probably due to neglect of greater than two-body interactions in the statistical-thermodynamic treatment. 2. In collaboration with the group of E. Haas we are studying the effect of a small "inert" cosolute, trimethylamine oxide, on the conformational isomerization of an enzyme, adenylate kinase, that is known to undergo significant conformational changes during its catalytic cycle. Conformational changes are being characterized via time-dependent fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores placed at selected locations along the peptide chain through site-specific mutagenesis. Results obtained to date indicate that sufficient concentrations of TMAO, in combination with selected ligands (ATP and AP4A, a transition state analog) can substantially bias conformational equilibria toward more compact conformations, in semi-quantitative agreement with predictions of excluded volume theory. 3. We are studying self-interaction of a synthetic polysaccharide (Ficoll 70) at high concentration and interaction between Ficoll 70 and tracer proteins (hemoglobin and BSA) at high Ficoll 70 concentration via a combination of composition-gradient light scattering and non-ideal tracer sedimentation equilibrium, two techniques developed previously in this laboratory. The results will provide the first comprehensive data on the thermodynamic interaction between proteins and polymers over a broad range of concentration, and provide essential information for the interpretation of a variety of crowding effects observed in the presence of high Ficoll concentrations (with A. Fodeke, C. Fernandez).