Noncovalent Intermolecular Interactions In Biochemistry

我们继续我们的高浓度的“惰性”大分子的影响，建模拥挤的细胞内环境，大分子协会和构象变化的平衡和动力学的研究。1.与M合作。Fried（Pennsylvania州立大学），一年前开始的关于高浓度“惰性”蛋白质对稀释的CAP（环AMP结合蛋白）和带有CAP特异位点的寡核苷酸结合的影响的凝胶转移研究仍在继续。2.一个新的简化分析示踪剂沉降平衡实验进行了高度非理想的解决方案已经开发和测试。该分析，检测和定量的可逆形成的标记示踪剂和其他未标记的大分子溶质在溶液混合物之间的复合物，是强大的，不需要任何有疑问的假设弱排斥力的性质大分子溶质之间的拥挤的解决方案。3.细胞色素c（cytc）和二油基磷酸甘油（DOPG）双层膜之间的相互作用正在通过几种技术进行研究。cytc和DOPG囊泡之间的可逆关联似乎是合作的，并与囊泡的融合。我们目前正试图通过低温电子显微镜和原子力显微镜研究这种联系的机制。4.建立了高浓度惰性大分子溶质（大分子拥挤）对成核聚合形成蛋白质纤维动力学影响的理论模型。动力学模拟使用各种各样的输入假设，某些结果似乎是定性独立于这些假设。拥挤被发现加速纤维形成的速率多达几个数量级，和拥挤引起的加速的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的影响的研究仍在继续。与Yisheng Ni（Courtesy Associates）合作，我们最近发现高浓度的葡聚糖在pH 2.0下稳定细胞色素c的熔融球构象，防止在高温和低温下展开，我们目前正在定量这种现象的能量学。6.通过荧光共振能量转移（FRET）测量，我们正在研究高浓度的葡聚糖对未折叠腺苷酸激酶构象的影响，以及天然蛋白质的“开放”和“封闭”构象之间的配体连接平衡，与Elisha哈斯（Bar-Ilan大学）合作。7.我们正在使用多组分理论的瑞利光散射在这个实验室开发的去年解释的数据，也获得了去年，在含有高达50克/升的葡聚糖的水溶剂中的选定的蛋白质的过量散射。初步结果表明，每个示踪蛋白质和葡聚糖之间的排斥相互作用，可以可靠地量化通过这样的测量。8.建立了蛋白质棒状聚合物溶液浊度随时间和波长变化的模型。它被用来分析在我们的实验室进行的时间和波长依赖性的微管蛋白进行聚合，形成微管在紫杉醇的存在下的溶液的浊度的测量结果。9.与German里瓦斯（生物研究中心）合作，我们正在继续对含有血浆主要蛋白组分和所有小分子组分的类血浆溶液中血浆蛋白的行为和相互作用进行沉降平衡研究。

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.