Noncovalent Intermolecular Interactions In Biochemistry

在与M.Fry(宾夕法尼亚州立大学)的合作下，一年前开始的关于高浓度“惰性”蛋白质对稀释型CAP(环磷酸腺苷结合蛋白)与带有CAP特定位点的寡核苷酸关联的影响的凝胶移位研究仍在继续。初步结果表明，高浓度名义上的惰性共溶体(肌红蛋白)的存在强烈促进了CAP与DNA的结合，但这种影响的量化进展比预期的要慢，这是因为Fry博士和他的整个研究小组在2004年春和夏迁往肯塔基大学。 与马德里生物调查中心(G.Rivas)的实验室合作，发展了一套完整的沉淀平衡理论，适用于任意浓度下的任意数量的溶质，并应用于分析浓度高达150g/L的核糖核酸酶溶液的行为。在较高浓度下，由于溶质物种之间排除的体积相互作用，加上形成小寡聚物(二聚体、三聚体、四聚体)的弱自缔合，核糖核酸酶溶液表现出极端的非理想性。 与P.McPhie(NIDDK-NIH)合作，在不同浓度的结构诱导盐三氯乙酸酯存在下，通过圆二色谱测量了高浓度葡聚糖对pH为2的熔融球状细胞色素c在冷热诱导下展开的影响。与排除体积理论的预测定性一致，发现高浓度的葡聚糖在冷诱导和热诱导的展开过程中都能稳定熔融球体。 关于惰性共溶质对天然蛋白质的稳定性和未折叠蛋白质构象的影响的新理论已经发展起来，取代了最初在本实验室发展并于2000年发表的理论。新的理论结合了分子内和分子间排除体积的更现实的处理方法，数值预测被发现与几个已发表的实验结果半定量或定性地一致。 继续与G.Rivas实验室合作，研究在含有与血浆中白蛋白和免疫球蛋白浓度相当的白蛋白和免疫球蛋白浓度的溶液中血浆蛋白的自相关和异类关联(CIB-CSIC，马德里)。 我们之前报道的细胞色素c与磷脂膜相互作用的研究被J.Hinshaw(NIDDK-NIH)获得的冷冻电子显微镜照片所加强，表明细胞色素c促进DOPG囊泡的融合，这些囊泡形成的聚集体取决于细胞色素c与DOPG的比例。通过与E.Dimitriadis(OD-NIH)合作，通过原子力显微摄影术将该蛋白质吸附到支撑的平面DOPG双层上的尝试被证明是不成功的。 我们已经开发了一种连续流动系统，用于测量溶液的随时间变化的光散射，该溶液的组成是以受控和已知的方式作为时间的函数变化的。数据是快速获得的，并且在非缔合和自缔合和异缔合大分子混合物的光散射模型的背景下被同样快速地分析。验证实验表明，该方法具有较高的准确度和精密度，可同时测定自缔合和杂缔合的分子量和平衡常数。 与D.Hall(剑桥大学)合作开发了一种蛋白质溶液的浑浊度理论，该蛋白质正在聚集形成棒状聚合物。该理论表明，基于浊度与蛋白质聚集量成正比的假设，对单波长实验确定的随时间变化的浊度的传统解释，在短时间和短棒长时是不准确的。该理论表明，浊度的波长相关性可以用来确定在单一波长观察到的浊度是更合适地解释为衡量加权平均聚合度还是更合适地解释为形成聚合物的总量。

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.