Noncovalent Intermolecular Interactions In Biochemistry

我们继续研究高浓度“惰性”大分子的影响，模拟拥挤的细胞内环境，基于大分子结合和构象变化的平衡和动力学。1.与M.Fry(宾夕法尼亚州立大学)合作，继续进行凝胶移位研究，研究高浓度的“惰性”蛋白质对稀释型CAP(环腺苷结合蛋白)与一年前开始的带有CAP特定位点的寡核苷酸之间的联系的影响。2.建立并验证了在高度非理想溶液中进行示踪剂沉降平衡实验的新简化分析方法。这种分析检测和定量标记示踪剂和溶液混合物中其他未标记大分子溶质之间的可逆形成的络合物，是稳健的，并且不需要关于拥挤溶液中大分子溶质之间作用的弱排斥力的性质的可疑假设。3.细胞色素c(Cytc)与双油基磷甘油(DOPG)双层膜的相互作用正在用多种方法研究。Cytc和DOPG囊泡之间的可逆结合似乎是合作的，并与囊泡的融合有关。我们目前正试图通过低温电子显微镜和原子力显微镜来研究这种连接的机制。4.建立了高浓度惰性大分子溶质(大分子拥挤)对蛋白质纤维成核聚合动力学影响的理论模型。动力学模拟使用了各种各样的输入假设，某些结果似乎定性地独立于这些假设。研究发现，拥挤使纤维的形成速度加快了几个数量级，而且拥挤引起的加速程度敏感地取决于聚合物核的大小。5.惰性大分子溶质对蛋白质稳定性和构象影响的研究仍在继续。最近，我们与益生(礼仪协会)合作，发现高浓度的葡聚糖稳定了pH为2.0的细胞色素c熔融的球状构象，使其在高温和低温下都不会展开，目前我们正在对这种现象的能量学进行量化。6.通过荧光共振能量转移(FRET)测量，我们与巴伊兰大学的Elisha Haas合作，研究了高浓度的葡聚糖对未折叠的腺苷酸激酶构象的影响，以及对天然蛋白质的“开放”构象和“封闭”构象之间的配基连接平衡的影响。7.我们正在使用本实验室去年开发的瑞利光散射多组分理论来解释去年也获得的数据，这些数据涉及选定的蛋白质在含有高达50克/L的葡聚糖的水溶液中的过度散射。初步结果表明，通过这样的测量，可以可靠地量化每个示踪蛋白与葡聚糖之间的排斥相互作用。8.建立了蛋白质形成棒状聚合物溶液中浊度随时间和波长变化的模型。它被用来分析我们实验室对在紫杉醇存在下聚合形成微管的微管蛋白溶液随时间和波长变化的浊度的测量结果。9.与德国生物调查中心合作，我们正在继续对血浆蛋白质在含有血浆主要蛋白质成分和所有小分子成分的类似血浆的溶液中的行为和相互作用进行沉淀平衡研究。

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.