DIRECT MEASUREMENT AND COMPUTATION OF FORCES BETWEEN MEMBRANES OR MACROMOLECULES

分子间的物理作用力将它们的结构与生物上重要的复合体的功能联系在一起。它们使我们能够预测蛋白质、核酸、脂双层和碳水化合物之间相互作用的强度和特异性。我们发现，水的结构在迄今研究的所有生物系统的密切相互作用中起着出乎意料的大作用。我们已经能够使用渗透胁迫方法和竞争平衡结合分析来测量从特定DNA结合蛋白EcoRI和非同源DNA序列之间的复合体中去除水分所需的工作。我们的策略是将宏观凝聚阵列中分子之间的力测量与稀溶液中分子的相互作用相结合。我们对EcoRI与其他非特异性DNA序列的复合体的结果检测了渗透胁迫下解离常数的变化。现在我们已经能够确定特定络合物离解率的离解率。该速率随渗透胁迫线性减慢，对各种大小和化学性质的溶质同一性不敏感。解离的渗透敏感性实际上与EcoRI特异和非特异性结合模式的渗透敏感性的差异是相同的。结果表明：a)解离速率可以用来代替平衡分析来测量被EcoRI-DNA复合体隔离的水的变化(以允许我们使用更高的渗透压力，而不会出现伴随平衡测量的并发症；b)正是脱水的、特定的相关状态通过渗透压力或拥挤来稳定。我们量化了小溶质与凝聚在有序宏观阵列中的大分子表面的相互作用。从这些小溶质对直接测量的DNA双螺旋之间和多糖之间的作用力的影响，我们发现小分子的溶解度随离大分子表面的距离呈指数变化。这已经在Hofmeister系列中的几种盐和几种已知可以稳定天然蛋白质结构的中性多元或两性离子溶质中看到。盐之间相互作用的大小与它们对水结构的已知影响相关，进一步表明水结构作用力对溶液中分子相互作用的重要性和普遍性。与此同时，我们一直在对聚电解质进行理论物理研究，特别是DNA。我们已经证明，在病毒中发现的致密密度下，DNA包装在液晶中，其角序是晶体的角序，但其间距像液体一样无序。我们已经开发了DNA组装的状态方程，以便将测量或计算的力与分子堆积的方式联系起来。当长DNA分子弯曲接触时，分子之间的相同相互作用也被应用于阐明它们的机械稳定性。这些力量也在DNA与阳离子脂类的组装过程中发挥作用，这些阳离子脂类用于准备导入。我们与NASA的合作现在已经发展到了这样一个地步，我们正在安装一种能够将光子流量提高10到100倍的X射线透镜。我们将在我们的力测量中使用这种新透镜，并测试X射线衍射的新策略。-水，渗透胁迫。蛋白质/DNA复合体，碳水化合物，DNA，分子作用力，X射线衍射。

Physical forces between molecules link their structure with the function of biologically important complexes. They let us predict the strength and specificity of interactions among proteins, nucleic acids, lipid bilayers, and carbohydrates. We have found that water structuring plays an unexpectedly large role in the close interaction of all biological systems so far investigated. We have been able to follow up our measurement of the work required to remove water from a complex between the specific DNA binding protein EcoRI and a non-cognate DNA sequence using the osmotic stress approach and a competitive equilibrium binding assay. Our strategy is to couple force measurements between molecules in macroscopic condensed arrays with the interaction of molecules in dilute solution. Our results on complexes of EcoRI with other, nonspecific DNA sequences examined changes in dissociation constant under the osmotic stress. Now we have been able to determine the dissociation rate of the specific complex dissociation rate. The rate slows linearly with osmotic stress and is insensitive to solute identity for a wide range of sizes and chemical natures. The osmotic sensitivity of dissociation is virtually identical to the difference in osmotic sensitivity of specific and nonspecific binding modes of EcoRI. It emerges that: a) Dissociation rates can be used instead of equilibrium assays to measure changes in water sequestered by EcoRI-DNA complexes (to allow us to use much higher osmotic stresses without the complications accompanying equilibrium measurements; b) It is the dehydrated, specifically associated state that is stabilized by osmotic stress or crowding. We have quantified the interaction of small solutes with macromolecular surfaces condensed in ordered macroscopic arrays. From the effect of these small solutes on directly measured forces between DNA double helices and between polysaccharides, we have found that small-molecule solubility varies exponentially with the distance from the macromolecular surface. This has been seen with several salts in the Hofmeister series and several neutral polyhydric or zwitterionic solutes that are known to stabilize native protein structures. The magnitude of the interaction of salts correlates with their known effect on water structure, further showing the importance and ubiquity of water structuring forces on the interaction of molecules in solution. At the same time we have been doing theoretical physical studies on polyelectrolytes, particularly DNA. We have demonstrated that at the compact densities found in viruses, DNA packs in a liquid-crystal whose angular order is that of a crystal but whose spacings are disorderly like those of a liquid. We have developed an equation of state for DNA assembly in order to relate measured or computed forces to the ways in which the molecules pack. The same interactions between molecules have also been applied to formulate the mechanical stability of long DNA molecules when they bend to come into contact. These forces come into play also in the assembly of DNA with cationic lipids as used in preparations for transfection. Our association with NASA has now developed to the point where we are installing an x-ray lens able to boost photon flux by a factor of 10 to 100 times. We will be using this new lens in our force measurements as well as testing new strategies for x-ray diffraction. - Water, Osmotic stress. Protein/DNA complex, carbohydrate, DNA, molecular forces, x-ray diffraction.