INTERACTIONS OF SEQUENCE-GENERAL DNA-BINDING PROTEINS

一般序列 DNA 结合蛋白的相互作用

• 批准号: 2688129
• 负责人: ANDREW H WANG
• 金额: $ 22.75万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2688129
• 关键词: Archaea; DNA binding protein; Escherichia coli; X ray crystallography; bacterial proteins; bacteriophage M13; chemical stability; circular dichroism; conformation; electron microscopy; histones; intermolecular interaction; mass spectrometry; molecular polarity; molecular site; mutant; nuclear magnetic resonance spectroscopy; nucleic acid structure; oligonucleotides; protein binding; protein sequence; site directed mutagenesis; structural biology; virus protein

DESCRIPTION: The overall aim is to investigate the molecular basis of protein-DNA interactions through the high resolution crystal structural analysis of a number of sequence-general DNA-binding proteins and their complexes with DNA. In particular, chromosomal proteins from archaeabacteria (Sulfolobus) and bacteria (E. coli) complexed to double-stranded DNA and bacteriophage single-stranded DNA binding proteins complexed to ssDNA will be the major focus. Specific Aim I. Double-stranded DNA-binding proteins. (A) Sac7d and Sso7d are two dsDNA-binding proteins that play the important role of stabilizing dsDNA in hyperthermophile archabacteria. Dr. Wang has obtained high quality crystals of Sac7d and Sso7d bound to DNA (resolution between 1.3 Angstrom to 1.9 Angstrom). He will study: (1) the novel DNA-binding mode of Sac7d/Sso7d through the structural analysis of the protein-DNA complexes, (2) the conformational modulation of DNA at the kink site, (3) the conformational modulation of proteins using site-directed mutants of Sac7d/Ddo7d. (B) HU protein from E. coli is a bacterial chromatin protein that forms a "nucleosome-like" structure. Dr. Wang has obtained diffraction-quality crystals of HU protein bound to a DNA octanucleotide. He proposes to solve the structure of this HU-DNA complex to its highest resolution possible. Comparison of chromosomal proteins from archaea (Sac7d, HMfb), bacterial (HU) and eucaryotic (histones) kingdoms will be useful in the understanding of the evolution of chromatin organization. Specific Aim II. Single-stranded DNA-binding proteins are important in the life cycle of many cells. Dr. Wang has determined the 1.6 Angstrom resolution structure of the M13 bacteriophage gene V protein (GVP) and proposed a model for its superheical protein-DNA complex. He will determine the crystal structure of the M13 GVP complexed with DNA hexamers which will allow a direct visualization of the protein-DNA interaction. In addition, the GVP from another I2-2 phage (108 a.a) will be analyzed at better than 2.0 Angstrom resolution. The above protein-DNA systems will be studied by cryo x-ray crystallography and their structures correlated with biological functions. Other biophysical techniques will also be employed when needed. These studies will greatly improve our understanding on the biological functions of those sequence-general DNA-binding proteins.

描述：总体目标是研究分子基础 通过高分辨率晶体结构的蛋白质-DNA 相互作用 一些序列通用 DNA 结合蛋白的分析及其 与DNA形成复合物。 特别是，染色体蛋白来自 古细菌（硫化叶菌）和细菌（大肠杆菌）复合 双链DNA和噬菌体单链DNA结合蛋白 与 ssDNA 复合将是主要焦点。 具体目标 I. 双链 DNA 结合蛋白。 (A) Sac7d 和 Sso7d 是两种 dsDNA 结合蛋白，它们在稳定 超嗜热古细菌中的双链 DNA。 王博士获得高品质 Sac7d 和 Sso7d 晶体与 DNA 结合（分辨率在 1.3 埃到 1.9埃）。 他将研究：（1）新型DNA结合模式 Sac7d/Sso7d 通过蛋白质-DNA复合物的结构分析， (2) DNA 在扭结位点的构象调节，(3) 使用定点突变体进行蛋白质构象调节 Sac7d/Ddo7d。 (B) 来自大肠杆菌的 HU 蛋白是一种细菌染色质蛋白 形成“核小体样”结构。 王博士获得 与 DNA 八核苷酸结合的 HU 蛋白的衍射质量晶体。 他提议将这种 HU-DNA 复合物的结构解析到最高水平 分辨率可能。 古细菌染色体蛋白的比较 （Sac7d、HMfb）、细菌（HU）和真核（组蛋白）王国将 有助于理解染色质组织的进化。 具体目标二。 单链 DNA 结合蛋白在 许多细胞的生命周期。 王博士测定了1.6埃 M13噬菌体基因V蛋白（GVP）的解析结构和 提出了其超螺旋蛋白质-DNA 复合物的模型。 他将决定 M13 GVP 与 DNA 六聚体复合的晶体结构 允许蛋白质-DNA 相互作用的直接可视化。 此外， 来自另一个 I2-2 噬菌体 (108 a.a) 的 GVP 的分析结果将优于 2.0 埃分辨率。 上述蛋白质-DNA系统将通过冷冻X射线晶体学进行研究 及其结构与生物功能的相关性。 其他 需要时还将采用生物物理技术。 这些研究 将极大地提高我们对这些生物功能的理解 序列通用 DNA 结合蛋白。