QUANTITATIVE IN VIVO/IN VITRO STUDIES OF CELL PROCESSES

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DESCRIPTION: The two long term goals of this research are: 1) To obtain quantitative in vivo-in vitro relationships between biopolymer processes, which occur in the concentrated, multi-solution of only a few components, and 2) To obtain a quantitative characterization of the model organism E. coli as an adaptable chemical and osmotic system. These two long-term goals are closely related. In E. coli, biopolymer interactions occur in a highly crowded, polyelectrolyte environment, where amounts of water and of ionic and nonionic solutes change in response to changes in the surroundings (e.g. changes in osmolarity of the growth medium). Macromolecular crowding, polyelectrolyte effects, and preferential interactions or local concentrations of water, solutes, and macromolecular sites (all of which change with conditions of growth) must individually exert large and important effects on cellular processes involving biopolymers; these individual effects must be understood in order to compare in vitro and in vivo studies. To accomplish these long term goals, Dr. Record proposes as specific aims an interrelated set of quantitative in vitro and/or in vivo studies of ion-ribosome interactions, of interactions of water and cytoplasmic osmolytes (and related solutes) with proteins and nucleoproteins, and of effects of macromolecular crowding. He proposes to use NMR of quadrupolar ions, osmometry and equilibrium dialysis to quantify contributions to molecular and thermodynamic properties of model systems in vitro. The osmolarity and presence of osmoprotectants in the growth medium are reproducible, quantitative and independent variables that determine key thermodynamic properties of the cytoplasm of E. coli. Both the thermodynamic activity of water and the concentrations of various solutes (both polymeric and nonploymeric) in the cytoplasm can be changed in a reproducible manner by changing the osmolarity and composition of the growth medium. An understanding at the biophysical chemical level of the relationship between environmental influences and cellular processes in this chemically complex but biologically well-characterized organism (E. coli) is of fundamental biomedical relevance.