Direct Measurement and Computation of Forces Between Membranes or Macromolecules

分子之间的物理作用力将它们的结构与生物学上重要的复合物的功能联系起来。它们让我们预测蛋白质、核酸、脂质双分子层和碳水化合物之间相互作用的强度和特异性。我们发现，水结构在所有生物系统的密切相互作用中起着意想不到的重要作用。我们已经能够使用渗透胁迫方法和竞争平衡结合试验来跟踪测量从特定DNA结合蛋白EcoRI和非同源DNA序列之间的复合物中去除水所需的工作。我们的策略是将宏观凝聚态阵列中分子间的力测量与稀溶液中分子间的相互作用耦合起来。我们对EcoRI与其他非特异性DNA序列复合物的研究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的解离速率。速率随渗透应力线性减慢，对溶质同一性不敏感，适用于各种尺寸和化学性质。解离的渗透敏感性实际上与EcoRI特异性和非特异性结合模式的渗透敏感性差异相同。这表明：a)解离率可以代替平衡分析来测量EcoRI-DNA复合物所隔离的水的变化（允许我们使用更高的渗透应力，而没有伴随平衡测量的并发症；b）它是脱水的，特别是相关的状态，通过渗透压力或“拥挤”来稳定。我们已经量化了小溶质与大分子表面在有序宏观阵列中的相互作用。从这些小溶质对DNA双螺旋之间和多糖之间的直接测量力的影响来看，我们发现小分子的溶解度随着与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稳定天然蛋白质结构的中性多水合或两性离子溶质中看到。盐相互作用的大小与其对水结构的已知影响有关，进一步表明水结构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，特别是DNA进行理论物理研究。我们已经能够测试流行的DNA“离子凝聚”理论，并表明它无法定性地解释DNA渗透压。我们与美国国家航空航天局的合作现在已经发展到我们正在安装一个x射线透镜的地步，据说它可以将光子通量提高10到100倍。我们将在我们的力测量中使用这种新的透镜，并测试x射线衍射的新策略。

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 been able to test the popular 'ion-condensation' theory of DNA and have shown that it fails qualitatively to account for DNA osmotic pressures. Our association with NASA has now developed to the point where we are installing an x-ray lens said 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.