NEW PROBES AND REAGENTS FOR BIOLOGICAL STUDIES

该研究项目的中心主题是设计，化学 新型探针的合成、表征和协同应用 和研究生物系统的试剂。 靶分子 针对研究人员当前的需求和局限性而设计 以及生物化学、分子生物学、细胞生物学、 生物学和医学。 五个分领域构成了拟议的重点 五年计划。 a）新型光化学触发交联 试剂 交联剂提供了有价值的结构和性能。 近邻分子水平的机理信息 两个生物分子之间的相互作用，并且可以例如提供 理解复杂生物分子组装体功能的基础。 开发了一类新的氟化的、可裂解的、光化学的 预期具有上级性能的触发交联剂是 提出了 这些分子将被用于一项合作研究与博士。 Capaldi研究的重要酶的作用机制 F（o）F（1）-ATP合酶。 B）疏水腔内的光标记 突变的T4溶菌酶 光标记是最有效的方法之一 以获得小分子的蛋白质结合位点的信息。 博士马修斯最近描述了一系列新颖的晶体 突变的T4-溶菌酶，其具有良好限定的疏水腔， 小分子。 这些晶体为研究 新的光标记试剂的行为，因为它们占据了一个 疏水性“结合位点。“c）具有新的和不寻常的基质 生物化学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 在细胞功能中发挥核心作用，例如， 锚定在细胞表面的蛋白质或细胞增殖 信号，如激素分子在细胞表面的结合。 为了帮助合作的生物化学和生物物理研究， 细菌和哺乳动物的酶与格里菲斯博士，新的底物和 将开发抑制剂。 d）设计用于辅助 在原子上的水下单个DNA分子的可视化 原子力显微镜（AFM）。 的可视化 通过强大的成像技术对单个生物分子进行成像， 现实 然而，只有原子力显微镜似乎有可能观察到 生物分子在水环境中以其天然状态存在。 博士 布斯塔曼特最近发表了一组DNA分子的图像 在空气或丙醇-水下放置在（原子级平坦的）云母表面上。 由于DNA的运动，在水中成像是不可能的。 小说 将DNA在水下固定在云母表面的方法有 提出了 e）肌醇磷酸的微生物传感器。 酶偶联 场效应晶体管（ENFET）正经历快速发展， 高选择性和灵敏度的微生物传感器， 细胞内应用。 怀伯恩医生和首席调查员 最近已经将酶固定在制造的亚微米结构上 在怀伯恩博士的微电子学中使用电子束光刻技术 实验室 我们的目标是建立一个ENFET，将检测肌醇 磷酸盐，PI-PLC作用于磷酸二氢根的水解产物 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.