NEW PROBES AND REAGENTS FOR BIOLOGICAL STUDIES

这个研究项目的中心主题是设计，化学 新型探针的合成、表征及协同应用 以及研究生物系统的试剂。目标分子是 针对研究人员的当前需求和限制而设计 和生物化学、分子生物学、细胞等领域的从业者 生物和医学。五个子领域构成了本建议的重点 五年计划。A)新颖的光化学触发的交联剂 试剂。交联剂提供了有价值的结构和 分子水平上关于近邻的机理信息 两个生物分子之间的相互作用，并且例如可以提供 了解复杂的生物分子组件如何发挥作用的基础。 一类新型氟化、可裂解光化学材料的研制 有望具有优异性能的触发交联剂是 建议。这些分子将用于与Dr。 卡帕尔迪研究重要酶的作用机制 F(O)F(1)-三磷酸腺苷合成酶。B)疏水空腔内的光标记 突变的T4-溶菌酶。光标记是最有成效的方法之一 以获得有关小分子的蛋白质结合部位的信息。 马修斯博士最近描述了一系列新的水晶 突变的T4-溶菌酶具有明确的疏水空腔，可结合 小分子。这些晶体为研究提供了一个独特的机会 当新的光标记试剂占据一个 疏水的“结合部位”。C)具有新的和不同寻常的衬底 用于生化和生物物理研究的性质 磷脂酰肌醇特异性磷脂酶C(PI-PLC)。PI-PLC酶 在细胞功能中占据中心角色，例如，释放 锚定在细胞表面的蛋白质或细胞的放大 信号，如激素分子与细胞表面的结合。 协助合作进行生物化学和生物物理研究 与格里菲斯博士合作的细菌和哺乳动物酶，新的底物和 将开发出抑制剂。D)新型试剂，旨在帮助 水下单个DNA分子在原子计算机上的可视化 原子力显微镜(AFM)观察表面平整。视觉化的 单个生物分子通过强大的成像技术正在成为一种 现实。然而，似乎只有原子力显微镜才有可能观察到 水环境中处于天然状态的生物分子。Dr。 布斯塔曼特最近发表了单个DNA分子的图像 躺在空气或丙醇-水下的(原子平坦的)云母表面上。 由于DNA的运动，在水中成像是不可能的。小说 将水下DNA固定在云母表面的方法有 建议。E)肌醇磷酸的微生物传感器。酶偶联 场效应晶体管(ENFET)正经历着快速发展 具有高选择性和高灵敏度的微生物传感器 细胞内应用。怀伯恩博士和首席调查员 最近在制造的亚微米结构上固定酶 在怀伯恩博士的微电子学中使用电子束光刻 实验室。目标是建立一种能检测肌醇的ENFET 磷酸盐，PI-PLC作用于磷二酰基的水解产物 肌醇。

The central theme of this research project is the design, chemical synthesis, characterization and collaborative application of new probes and reagents for studying biological systems. Target molecules are designed in response to the current needs and limitations of researchers and practitioners in the fields of biochemistry, molecular biology, cell biology and medicine. Five sub-areas form the focus of this proposed five-year project. a) Novel photochemically triggered cross-linking reagents. Cross-linking reagents provide valuable structural and mechanistic information at the molecular level about near-neighbor interactions between two biomolecules and may, for example, provide a basis for understanding how complex biomolecular assemblies function. The development of a new class of fluorinated, cleavable, photochemically triggered cross-linking reagents expected to have superior properties is proposed. These molecules will be used in a collaborative study with Dr. Capaldi to study the mechanism of action of the important enzyme F(o)F(1)-ATP synthase. b) Photolabeling within the hydrophobic cavity of mutated T4-lysozyme. Photolabeling is one of the most fruitful ways to gain information about the protein binding site of small molecules. Dr. Matthews has very recently described a novel series of crystalline mutated T4-lysozymes having a well defined hydrophobic cavity that binds small molecules. The crystals provide a unique opportunity for studying the behavior of the new photolabeling reagents as they occupy a hydrophobic "binding site." c) Substrates with new and unusual properties for biochemical and biophysical studies of phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC enzymes occupy a central role in cellular function, for example, the release of proteins anchored to the cell surface or the amplification of cellular signals such as the binding of a hormone molecule on the cell surface. To aid in collaborative biochemical and biophysical studies of the bacterial and mammalian enzymes with Dr. Griffith, novel substrates and inhibitors will be developed. d) Novel reagents designed to aid in the visualization of individual DNA molecules under water on an atomically flat surface by atomic force microscopy (AFM). The visualization of individual biomolecules by powerful imaging techniques is becoming a reality. However, only AFM appears to have, the potential to observe biomolecules in an aqueous environment in their native state. Dr. Bustamante has very recently published images of individual DNA molecules laying on a (atomically flat) mica surface under air or propanol-water. Imaging in water was not possible owing to motion of the DNA. Novel approaches toward pinning the DNA under water to the mica surface are proposed. e) A microbiosensor for inositol phosphate. Enzyme coupled field effect transistors (ENFETs) are undergoing rapid development as highly selective and sensitive microbiosensors with potential for intracellular applications. Dr. Wybourne and the principal investigator have recently immobilized enzymes on sub-micron structures fabricated with electron-beam lithography in Dr. Wybourne's microelectronics laboratory. The objective is to build an ENFET that will detect inositol phosphate, the hydrolysis product of the action PI-PLC on phosphadityl inositol.