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 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. A report of this work has been published. 2. The sedimentation equilibrium of concentrated Ficoll 70 and dilute labeled BSA in Ficoll 70 has been measured as a function of Ficoll 70 concentration up to 100 g/l. The sedimentation equilibrium of concentrated BSA and dilute labeled Ficoll 708 has been measured as a function of BSA concentration up to 100 g/l. The results have been analyzed in the context of a general thermodynamic description of Ficoll - Ficoll, BSA - BSA, and Ficoll-BSA intermolecular interactions. The results have also been analyzed in the context of an equivalent hard particle model description. A report of this work has been published. 3. 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. This work is being written up for publication. 4. In collaboration with the group of Y. Liang, the association of catalase and superoxide dismutase was measured by fluorescence titration as a function of the concentration of each of three different polymers (dextran 70, ficoll 70, PEG 10K) and temperature. At high temperatures the addition of each polymer was found to strongly enhance association of the two proteins, but at low temperature (ca 7 deg C) the addition of polymer had little or no effect on the association. The findings were interpreted in the context of a model, according to which attractive protein-polymer interactions inhibit protein association and repulsive protein-polymer interactions enhance protein association. The balance between these two effects is strongly temperature-dependent. A report of this work has been published.
