Studies of macromolecular crowding

1. 我们正试图改进我们先前发表的关于大分子拥挤对蛋白质结合和异构化影响的理论模型。目前的工作重点是在含有任意体积分数的“更硬”颗粒的流体中，两个“示踪”硬颗粒之间的平均力势的统计热力学公式。这不是一个可解析解决的问题，我们正在尝试各种近似技术，以达到一个健壮的解决方案，而不是严格依赖于模型公式中所作近似的性质。我们进行了计算，并将理论模型的结果与蒙特卡罗和/或分子动力学模拟（由N. Dokholyan）的结果进行了比较。结果表明，理论和模拟在示踪粒子之间的接触距离上是一致的，但在较大的粒子间距离上存在差异，这可能是由于统计热力学处理中忽略了大于两体的相互作用。

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).