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).

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2009
Total Cost
$261,638
Indirect Cost
City
State
Country
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Hoppe, Travis; Minton, Allen P (2015) An equilibrium model for the combined effect of macromolecular crowding and surface adsorption on the formation of linear protein fibrils. Biophys J 108:957-66
Beg, Ilyas; Minton, Allen P; Hassan, Imtaiyaz et al. (2015) Thermal Stabilization of Proteins by Mono- and Oligosaccharides: Measurement and Analysis in the Context of an Excluded Volume Model. Biochemistry 54:3594-603
Wu, Di; Minton, Allen P (2013) Compensating effects of urea and trimethylamine-N-oxide on the heteroassociation of ?-chymotrypsin and soybean trypsin inhibitor. J Phys Chem B 117:3554-9
Minton, Allen P (2013) Quantitative assessment of the relative contributions of steric repulsion and chemical interactions to macromolecular crowding. Biopolymers 99:239-44
Fernández, Cristina; Minton, Allen P (2011) Effect of nonadditive repulsive intermolecular interactions on the light scattering of concentrated protein-osmolyte mixtures. J Phys Chem B 115:1289-93
Nagarajan, Sureshbabu; Amir, Dan; Grupi, Asaf et al. (2011) Modulation of functionally significant conformational equilibria in adenylate kinase by high concentrations of trimethylamine oxide attributed to volume exclusion. Biophys J 100:2991-9
Fodeke, Adedayo A; Minton, Allen P (2010) Quantitative characterization of polymer-polymer, protein-protein, and polymer-protein interaction via tracer sedimentation equilibrium. J Phys Chem B 114:10876-80
Tsao, Douglas; Minton, Allen P; Dokholyan, Nikolay V (2010) A didactic model of macromolecular crowding effects on protein folding. PLoS One 5:e11936
Jiao, Ming; Li, Hong-Tao; Chen, Jie et al. (2010) Attractive protein-polymer interactions markedly alter the effect of macromolecular crowding on protein association equilibria. Biophys J 99:914-23
Zhou, Huan-Xiang; Rivas, German; Minton, Allen P (2008) Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu Rev Biophys 37:375-97