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. In collaboration with M. Fried (Pennsylvania State University), gel-shift studies 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 a year ago, are continuing. 2. A new simplified analysis of tracer sedimentation equilibrium experiments carried out in highly nonideal solutions has been developed and tested. The analysis, which detects and quantitates the reversible formation of complexes between a labeled tracer and other unlabeled macrosolutes in a solution mixture, is robust and requires no questionable assumptions regarding the nature of weak repulsive forces acting between macromolecular solutes in crowded solutions. 3. The interaction between cytochrome c (cytc) and dioleylphosphoglycerol (DOPG) bilayer membranes is being studied by several techniques. Reversible association between cytc and DOPG vesicles appears to be cooperative and linked to fusion of the vesicles. We are currently attempting to investigate the mechanism of this linkage via cryoelectron microscopy and atomic force microscopy. 4. A theoretical model for the effect of high concentrations of inert macrosolutes (macromolecular crowding) on the kinetics of protein fiber formation via nucleated polymerization has been developed. Kinetic simulations were performed using a broad variety of input assumptions, and certain results seem to be qualitatively independent of these assumptions. Crowding is found to accelerate the rate of fiber formation by as much as several orders of magnitude, and the degree of crowding-induced acceleration depends sensitively upon the size of the polymer nucleus. 5. Studies of the effect of inert macrosolutes on protein stability and conformation continue. In collaboration with Yisheng Ni (Courtesy Associates), we have recently found that high concentrations of dextran stabilize the molten globule conformation of cytochrome c at pH 2.0 against unfolding at both high and low temperature, and we are presently in the process of quantitating the energetics of this phenomenon. 6. By means of fluorescence resonance energy transfer (FRET) measurements, we are studying the effect of high concentrations of dextran on the conformation of unfolded adenylate kinase, and upon the ligand-linked equilibrium between """"""""open"""""""" and """"""""closed"""""""" conformations of the native protein, in collaboration with Elisha Haas (Bar-Ilan University). 7. We are using the multicomponent theory of Rayleigh light scattering developed in this lab last year to interpret data, also obtained last year, on the excess scattering of selected proteins in aqueous solvents containing up to 50 g/l of dextran. Preliminary results indicate that the repulsive interaction between each tracer protein and dextran may be reliably quantified by means of such measurements. 8. A model for time- and wavelength-dependence of turbidity in solutions of proteins forming rod-like polymers has been developed. It is being used to analyze the results of measurements performed in our lab of the time- and wavelength-dependent turbidity of solutions of tubulin undergoing polymerization to form microtubules in the presence of taxol. 9. In collaboration with German Rivas (Center for Biological Investigations) we are continuing sedimentation equilibrium studies of the behavior and interactions of plasma proteins in a plasma-like solution containing the major protein components and all of the small molecule components of blood plasma.
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