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. Intital results suggest that the presence of high concentrations of a nominally inert cosolute (myoglobin) strongly promotes the binding of CAP to DNA, but progress in quantification of this effect has been slower than anticipated, due to the relocation of Dr. Fried and his entire research group to the University of Kentucky during the spring and summer of 2004. In collaboration with the laboratory of G. Rivas (Center for Biological Investigations, Madrid), a complete theory of sedimentation equilibrium, applicable to an arbitrary number of solutes at arbitrary concentrations, has been developed and applied to the analysis of the behavior of ribonuclease solutions at concentrations of up to 150 g/l. At higher concentrations the ribonuclease solution exhibits extreme nonideality due to excluded volume interactions between solute species, coupled with weak self-association to form small oligomers (dimers, trimers, tetramers). In collaboration with P. McPhie (NIDDK-NIH), the effect of high concentrations of dextran upon the heat- and cold-induced unfolding of the molten globule form of cytochrome c at pH 2 was measured via circular dichroism spectroscopy in the presence of various concentrations of the structure-inducing salt trichloroacetate. In qualitative accord with predictions of excluded volume theory, high concentrations of dextran are found to stabilize the molten globule with respect to cold-induced as well as heat-induced unfolding. A new theory of the effect of inert cosolutes upon the stability of native proteins and the conformation of unfolded proteins has been developed, superseding the theory initially developed in this laboratory and published in 2000. The new theory incorporates significantly more realistic treatments of both intramolecular and intermolecular excluded volume, and numerical predictions are found to agree semiquantitatively or qualitatively with several published experimental findings. Studies of the self- and hetero-associations of plasma proteins in solutions containing concentrations of albumin and immunoglobulins comparable to their concentrations in blood plasma, carried out in collaboration with the laboratory of G. Rivas (CIB-CSIC, Madrid), continue. Our previously reported study of the interaction of cytochrome c with phospholipid membranes was augmented by cryo-electron micrographs obtained by J. Hinshaw (NIDDK-NIH), showing that cytochrome c facilitates fusion of DOPG vesicles, which form a variety of aggregates depending upon the ratio of cytochrome c to DOPG. Attempts to visualize the adsorption of this protein to a supported planar DOPG bilayer by means of atomic force micrography, in collaboration with E. Dimitriadis (OD-NIH), did not prove successful. We have developed a continuous flow system for measuring the time-dependent light scattering of a solution whose composition is being varied in a controlled and known manner as a function of time. Data are obtained rapidly, and are equally rapidly analyzed in the context of models for light scattering of mixtures of non-associating and self- and hetero-associating macromolecules. Validation experiments have shown that the method is capable of measuring both molecular weights and equilibrium constants for self- and heteroassociation with high accuracy and precision. A theory for turbidity of a solution of a protein that is aggregating to form rod-like polymers has been developed in collaboration with D. Hall (Cambridge Univ). The theory shows that the conventional interpretation of experimentally determined time-dependent turbidity at a single wavelength, based upon the assumption that turbidity is proportional to the amount of protein aggregated, is inaccurate at short times and short rod lengths. The theory suggests that the wavelength dependence of turbidity can be used to determine whether the turbidity observed at a single wavelength is more appropriately interpreted as a measure of the weight-average degree of polymerization or as a measure of the total amount of polymer formed.
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