The technique of nonideal tracer sedimentation equilibrium recently developed in our laboratory has been used to measure the effect of high concentrations (up to 150 g/l) of either of two proteins, hemoglobin (Hb) or bovine serum albumin (BSA), on the thermodynamic activity and/or state of association of the bacterial cell division protein FtsZ in the absence of Mg ion, where FtsZ does not self-associate, and in the presence of Mg, where FtsZ self-associates to form a heterogeneous mixture of linear rodlike oligomers (see last year's report). It was found that in the absence of Mg, the behavior of FtsZ in the presence of either Hb or BSA may be accounted for quantitatively by the assumption that interactions between monomeric FtsZ and either BSA or Hb are purely steric-repulsive, and may be quantitatively described by models in which each species is represented by an equivalent hard sphere with a size approximately equal to that of the actual protein molecule. It was also found that in the presence of Mg, the extent of FtsZ self-association for a given total concentration of FtsZ increases substantially with increasing concentration of either Hb or BSA in a manner that is quantitatively described by excluded volume theory for a model in which oligomers of FtsZ are represented by spherocylinders having a fixed radius approximately equal to that of monomeric FtsZ and a length determined by conservation of mass. The rates and equilibria governing the assembly of large arrays of tubulin were studied via measurement of the wavelength-dependent turbidity of tubulin solutions as a function of time and various experimental variables, including the concentration of protein, the concentration and type of guanine nucleotides, pH, buffer salts, temperature, and the concentration of various """"""""inert"""""""" additives. It is evident that addition of """"""""inert"""""""" additives greatly accelerates and increases the equilibrium extent of array formation in qualitative accord with predictions of excluded volume theory. Further experimentation and formulation of quantitative models of the assembly process are in progress. The formation of a specific complex between an DNA-binding protein, CAP, and a 42 base-pair oligonucleotide containing a sequence specific for CAP, has been studied by tracer sedimentation equilibrium in the absence and presence of high concentrations of """"""""inert"""""""" proteins in an attempt to discern the effect of the inert proteins on the affinity of CAP for the oligonucleotide. Quantitative models for this process are being formulated. The effect of dextran, an inert water-soluble polymer, on the rate of formation of amyloid fiber by apolipoprotein CII has been studied by time-dependent turbidimetry and time-dependent pelleting of polymer. It was found that addition of polymer at concentrations of up to 150 g/l increases the relative rate of amyloid formation by up to eight-fold in a manner that may be described quantitatively by a simple excluded volume model. The effect of dextran on the stability of lysozyme with respect to thermal denaturation has been studied via temperature dependence of absorbance and near and far uv circular dicrhroism. A small but significant increase in the half-denaturation temperature is found upon increasing dextran concentration to 300 g/l. Further studies aimed at elucidating the basis of this effect are underway.
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