DIRECT MEASUREMENT OF FORCES BETWEEN MEMBRANES OR MACROMOLECULES

我们测量分子间作用力的主题已经扩大了 今年，要看清特定与非特定的亲密关系 蛋白质/DNA，以及在阵列上看到的大规模干扰 被限制在墙之间的分子。我们的主要工具是 渗透胁迫的应用，系统变化的活性 水研究分子构象的变化和堆积耦合 相互作用和组装的物理学。 我们早期的研究表明，有更多的水被排出 调节蛋白和DNA之间的特定接触比与 非特定绑定。即使是结合的6个碱基对中的一个突变 限制性内切酶EcoRI削弱了结合强度和 残余分子间溶剂化为完全非特异性溶剂化 有约束力的。然而，鞋子中突变的鹅卵石的影响可能是 通过施加更高的渗透胁迫来缓解 特定结合的脱水。建立了一种竞争性结合分析方法 今年允许系统地调查两国之间的关系 分子结合的特异性、结合强度和持续时间。 用小角x射线检查了DNA-脂质凝聚体。 衍射揭示了带负电荷的DNA可以以多种方式堆积， 尤其是带正电的脂类。除了是一个模范系统 对于分子组装，这些凝聚物被用来促进DNA 转染法摄取。与这些研究同时进行的还有DNA研究。 在各种盐溶液中单独存在，其中液晶特性产生 根据DNA浓度和强度的不同而不同的填料 分子相互作用。 分子间力作用下DNA堆积的观察 与几个理论物理公式相结合 分子间的相互作用不仅随着分子间力的变化 不仅与距离有关，还与DNA轴之间的角度有关。这个 今年的进展是制定依赖于 聚集在DNA周围的离子的位置波动，In 特别是在DNA分子不需要相互作用时的演示 成对的。 一项大型研究已经开始，涉及五个不同的实验室使用相同的 脂类材料，建立了我们的强大的蒸汽压理论 “悖论”。膜和刚性大分子在以下情况下可以不同地包装 与宏观解决方案相比，它们被坚硬的墙包围。 墙造成的干扰可以穿透几毫米到一些 样本，当包装DNA或 膜有时必须在微米尺度内发生 一种病毒。 对堆积分子能量的相关研究使我们能够 为不同类型的双层的组织创建“相图” 多层阵列并开始测量结晶热 蛋白质的含量。希望必要的系统研究将 提供x射线测量所需的结晶策略 在结构上。

Our theme of measuring forces between molecules has been enlarged this year, to look down to the intimacies of specific vs. non-specific protein/DNA and up to the large-scale disturbances seen on arrays of molecules confined between walls. Our principal tool has been the application of osmotic stress, systematic variation of the activity of water to examine changes in molecular conformation and packing coupled with the physics of interaction and assembly. Our earlier studies had shown that there is more water expelled between regulatory protein and DNA making specific contact than with non-specific binding. Even one mutation in the 6 base pairs that bind the restriction endonuclease EcoRI weakens binding strength and residual intermolecular solvation to that of completely non-specific binding. However, the effect of the mutant pebble in the shoe can be mitigated by application of higher osmotic stress to reach the dehydration of specific binding. A competitive binding assay developed this year is allowing systematic investigation of the relation between specificity, binding strength and duration of molecular association. DNA-lipid condensates have been examined by small-angle x-ray diffraction to reveal many ways the negatively charged DNA can pack, particularly with positively charged lipids. Besides being a model system for molecular assembly, these condensates are used to facilitate DNA uptake in transfection. In parallel with these studies are those of DNA alone in various salt solutions where liquid-crystalline properties create various packing depending on DNA concentration and the strength of molecular interaction. These observations on DNA packing mediated by intermolecular forces are coupled with several theoretical physical formulations on the interaction between molecules not only as the intermolecular force varies with distance but also with the angle between the axes of the DNA. The advance this year was to formulate interactions that depend on the positional fluctuations of the ions that cluster around the DNA, in particular the demonstration when DNA molecules need not interact in pairs. A large study has begun, involve five different labs using the same lipid materials, built our theory of the powerful 'vapor pressure paradox'. Membranes and stiff macromolecules can pack differently when bounded by hard walls than when they are in macroscopic solutions. Disturbances created by the walls can penetrate millimeters into some samples, something to think about when the packing of DNA or membranes must sometimes take place within the micron dimensions of a virus. Related studies on the energy of packing molecules have allowed us to create 'phase diagrams' for the organization of bilayers in different multilayer arrays and to begin to measure the heats of crystallization of proteins. It is hoped that the necessary systematic studies will provide a strategy of crystallization necessary for x-ray determination of structure.