The long-term goal of our research is to understand the thermodynamic basis of cytoplasmic function. In particular, we focus on the massive intracellular nonideality due to polyelectrolyte effects (cation accumulation at the surface of nucleic acids), preferential interactions (accumulation/exclusion) of solutes with proteins, excluded volume effects (crowding) and solvent nonideality (macromolecular hydration). We seek to determine the key physical chemical properties that govern the equilibria and kinetics of noncovalent interactions of proteins and of nucleic acids in the cytoplasm. This research addresses directly the important problem of relating in vitro studies of the thermodynamics and kinetics of processes of biological molecules to their in vivo function. Only after the composition and relevant thermodynamic properties of the intracellular environment have been quantified will it be possible to simulate these properties reliably in vitro or to extrapolate correctly from measurements made in a dilute solution in vitro to the much more concentrated in vivo state. Motivated by the long-term goals summarized above, our specific aims encompass three tightly-interrelated areas: 1) obtaining the necessary data; 2) developing the relevant statistical thermodynamic framework in order to analyze these data and evaluate thermodynamic coefficients, and 3) using these thermodynamic coefficients to predict the consequences of extreme thermodynamic nonideality for cellular processes as a function of osmolarity. In particular, we are investigating the thermodynamic bases of a) an in vivo-in vitro paradox (first noted by us) regarding the absence of [salt]-effects on gene expression in vivo, b) the quantitative linkage (also first noted by us) between effects of osmolarity and osmoprotectants on growth rate, cytoplasmic K+ activity and cytoplasmic water volume, and c) the behavior of """"""""osmotic remedial"""""""" mutants, which constitute a large sub-class of conditional-lethal mutants in which the wild-type phenotype is restored by growth at high osmolarity. Our novel thermodynamic proposals to explain these and other aspects of cell function represent the first of what we expect to be numerous advances in the application of classical and statistical thermodynamics to the analysis of in vivo processes. Key physiological variables which we utilize to change the thermodynamic state of the cytoplasm are: a) external osmolarity and b) the presence or absence of """"""""osmoprotectants"""""""" (e.g. glycine betaine, proline) in the growth medium. To obtain thermodynamic information for cells and for cytoplasmic species, we will measure: a) volumes of cytoplasmic water in unplasmolyzed cells and in cells titrated with a plasmolyzing agent (e.g. NaCl); b) osmotic and turgor pressures, and osmotic coefficients of the cytoplasm; c) amounts, osmotic properties, and NMR characteristics of osmotically- regulated cytoplasmic species; d) amounts of electrolyte ions, oligocations and polyanions responsible for cytoplasmic electroneutrality; and e) amounts and states of association of cytoplasmic macromolecules responsible for crowding.
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