We continue our studies of the effect of high concentrations of """"""""inert"""""""" macromolecules, modeling the crowded intracellular environment, upon the equilibria and kinetics of macromolecular associations and conformational changes. 1. The study of the effect of high concentrations of an """"""""inert"""""""" protein upon the association of dilute CAP (cyclic AMP binding protein) and an oligonucleotide bearing a specific site for CAP begun last year is continuing. Additional data have been acquired from new sedimentation equilibrium experiments and from a new application of gel-shift electrophoresis. Preliminary analysis indicates a substantial enhancement of the affinity of CAP for the oligonucleotide in the presence of a high concentration of myoglobin. 2. The theory of sedimentation equilibrium in nonideal solutions has been generalized to treat the case of multiple concentrated species that may undergo reversible self- and/or hetero-association. The generalized theory has been applied to the analysis of the concentration-dependent behavior of ribonuclease A, which appears to be weakly self-associating at concentrations exceeding 30 mg/ml. 3. As reported last year, the temperature for half-denaturation of lysozyme at pH 2 was found to increase monotonically with increasing dextran. The precision of these measurements was increased and it was found that the enthalpy of unfolding is unaffected by dextran. Both of these observations agree qualitatively with predictions of a theoretical model for the effect of intermolecular excluded volume on protein stability. 4. The effect of dextran on the equilibrium between the fully unfolded and compact non-native """"""""molten globule"""""""" forms of cytochrome c at pH 2 was studied. It was found that addition of dextran substantially stabilized the more compact molten globule conformation relative to the unfolded conformation, and that the magnitude of the effect could be accurately predicted by a simple excluded volume model incorporating known structural information about the molecular sizes and shapes of the interacting species. 5. The theory of elastic (Rayleigh) light scattering in solution has been extended to treat the case of up to three interacting solute species at arbitrary concentration. 6. The effect of dextran on the rate and extent of association of GDP-tubulin has been studied by measuring the time and wavelength dependence of turbidity, and by measuring time-dependent Rayleight scattering of light at multiple scattering angles. Preliminary analysis of the results indicates that the addition of dextran stongly accelerates tubulin polymerization, in qualitative accordance with predictions of excluded volume theory.
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