Protein's behavior, such as enzyme activity, is a function of their thermodynamic activity and their state of hydration. Most studies of their behavior have been conducted in solution conditions far different from the intracellular environment which proteins exist. In the cytosol, protein concentration is high and the solution in state is markedly non-ideal. We plan to dissect the factors influencing these solution conditions, notably excluded volume and water activity, using a model of protein/PEG/water which we suggest closely approximates the solution conditions of the cytosol.
Our aim then is to study how these conditions influence protein-protein interaction, protein-ligand interaction and the conformational stability and motility of proteins. In preliminary studies, we have found that when two hemoglobins, in their deoxy states, are precipitated together, the solubility of the mixture is significantly higher than would be expected by the combined solubilities of the two components. A variety of purified proteins will be used and direct analysis of the solid and solution phases will be made. For some of the experiments we will use proteins tethered to gel particles by a 6-carbon spacer (in order to avoid problems due to protein aggregation). Techniques of analysis of the thermodynamics of protein-protein interaction and of conformational mobility which we will use will include microcalorimetric analysis, hydrogen exchange and the measurement of ligand-binding. Our experiments, by their nature, will require a relatively long term effort and thus we are projecting a 5 year study. Our approach will first address itself to the formation of a satisfactory base for subsequent studies. In this phase, we will characterize the physical properties of water/PEG and water/PEG/protein solutions with respect to H+ and H- activities, water activity (by vapor pressure measurements) and surface tension. In the second phase, which will be more in line with our ultimate aims, we will study: (a) the stability of proteins in the presence of PEG; (b) the formation of mixed solid states, including monomer-polymer assembly; and (c) protein-ligand interactions. All of these studies will be done under solution conditions approximating those of the cytosol, using the water/PEG/protein model system.
| Somogyi, B; Norman, J A; Zempel, L et al. (1988) Viscosity and transient solvent accessibility of Trp-63 in the native conformation of lysozyme. Biophys Chem 32:1-13 |
