EM PHYSICAL STUDIES OF DNA BINDING PROTEIN COMPLEXES

高分辨率电子显微镜用于物理研究， 蛋白质-核酸复合物在两个新领域的应用。 (1)第一个是 由类似但物理上不同的物质形成的复合物的比较 丝状病毒fd和Pfl的DNA结合蛋白; fd基因5 蛋白质是研究最深入的“模型”DNA结合蛋白质之一 Pfl类似物是最近发现的。 怎么 这些不同的蛋白质执行相似的功能？ 的详细 这些螺旋复合物的三维结构将是 研究，包括构象变化，拓扑重排， 解离和重组，以及可能的圆盘到螺旋的转变 伴随着从n=3到n=4结合模式的转变。 车型将 为蛋白质和DNA在这些复合物中的排列而开发， 蛋白质与DNA的相互作用，因为它们与生物功能有关。 （二） 研究的第二个领域涉及使用fd基因5蛋白作为一种 在多核苷酸上“包被”以扩增并直接可视化 多核苷酸螺旋结构，一种初步 调查显示，这是可行的。 这可以提供一种手段， 直接确认不寻常的结构，如左手“Z”或 “环出”形式的DNA，并与光谱数据的相关性表明， 这些DNA在溶液中的存在形式。 这项工作的方法主要涉及定量分析 负染色的核蛋白结构在电子显微镜下 显微镜在非常高的放大倍数，使用倾斜/旋转在一个 测角器阶段，以获得三维结构信息。 圆二色性（CD）光谱用于蛋白结合滴定 并关联溶液结构数据（例如，Z-DNA光谱）， 通过显微镜分析的结构。 这项工作是与健康有关的，因为它是（1）一项基础研究， 一类蛋白质（螺旋去稳定化蛋白质）的最佳可用实例是 蛋白质），可能普遍参与的过程， DNA复制、重组和修复，以及（2）异常DNA的研究 例如，可以进入转录控制的结构。 了解这些基本的生物过程对于 了解健康和疾病中的细胞功能。

High resolution electron microscopy is used in physical studies of protein-nucleic acid complexes in two new areas. (1) The first is a comparison of complexes formed by the analogous but physically dissimilar DNA binding proteins of the filamentous viruses fd and Pfl; the fd gene 5 protein is one of the most intensively studied "model" DNA binding proteins presently available, and the Pfl analogue was recently discovered. How do these dissimilar proteins perform similar functions? The detailed three-dimensional structure of these helical complexes will be investigated, including conformational shifts, topological rearrangements, dissociation and reassembly, and a possible disk-to-helix transition accompanying a shift from an n=3 to an n=4 binding mode. Models will be developed for the arrangement of proteins and DNA in these complexes and for protein-DNA interactions as they relate to biological function. (2) The second area of study involves the use of the fd gene 5 protein as a "coating" on polynucleotides to amplify and directly visualize polynucleotide helical structure, an approach which preliminary investigations indicate should be feasible. This may provide a means for direct confirmation of unusual structures such as the left-handed "Z" or "loopout" forms of DNA, and for correlation with spectral data indicating the existence of these DNA forms in solution. The methodology for this work primarily involves the quantitative analysis of negatively-stained nucleoprotein structures visualized in an electron microscope at very high magnifications, using tilting/rotation in a goniometer stage to derive three-dimensional structural information. Circular dichroism (CD) spectroscopy is used for protein binding titrations and to correlate solution structural data (e.g., Z-DNA spectrum) with structures analyzed by microscopy. This work is health-related in that it is (1) a fundamental study of one of the best available examples of a class of proteins (helix-destabilizing proteins) that are likely to be universally involved in the processes of DNA replication, recombination, and repair, and (2) a study of unusual DNA structures that may enter into, for example, transcriptional control. Knowledge of these elemental biological processes is essential to understanding cellular function in health and disease.