NMR STUDIES OF STRUCTURE AND DYNAMICS OF SKIN PROTEINS AND VIRAL CASPID PROTEINS

皮肤蛋白和病毒衣壳蛋白的结构和动力学的核磁共振研究

• 批准号: 6344838
• 负责人: JAMES W. MACK
• 金额: $ 8.82万
• 依托单位: HOWARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344838
• 关键词: DNA binding protein; animal tissue; conformation; cytoskeletal proteins; fibrous protein; gel mobility shift assay; homeobox genes; intermediate filaments; keratin; molecular dynamics; nuclear magnetic resonance spectroscopy; protein folding; protein structure function; pulsed field gel electrophoresis; skin; thymine; virus envelope; virus protein

Determination of the three-dimensional structure of an intact homeodomain protein, and probing of the internal molecular dynamics of duplexed DNA specifically bound to the homeobox segment of a related protein is proposed. The full-length protein chosen for structure determination is the 193-residue NKx2.8 (MW=21.9 kD), recently discovered as native to chicken heart, which is one of the smallest known members of the NK2 class of homeodomain proteins. Internal dynamics of the canonical NK2 class recognitions DNA sequence will be characterized in part, with the sequence complexed to the 80-residue vnd/NK-2 homeobox protein. Homeodomains proteins bear the 60-residue long homeobox DNA-binding sequence that seems highly conserved, evolutionarily, across all eukaryotic species including humans. They are sequence-specific DNA-binding factors that generally trigger the timing of gene activation in contexts of embryonic development under ordinary circumstances, thus play roles in inducing cell type discrimination, morphogenesis, and growth control. Further, chromosomal translocations that substitute homeoboxes for normally occurring DNA- binding segments of transcription regulators have been linked with oncogenic activation of certain proto-oncogenes (as in some T-cell lymphomas). No structure of an intact homeodomain protein exists as yet. We propose to use methods of high resolution nuclear magnetic resonance spectroscopy (NMR) to determine the solution structure of recombinantly generated NKx2.8 in its free state. In addition to the homeobox itself, NKx2.8 contains two other sub-sequences that have been evolutionarily conserved to a significant degree. These are the 12-residue TN domain, upstream of the homeobox near the N-terminus of the protein, and the 23- residue NK2-SD domain downstream of the homeobox towards the C-terminus. Results recently reported in the literature are only beginning to offer clues as to the functional significance of these domains. No structural data exists for either of these domains as yet. Thus, the development of a detailed description of the structure of NKx2.8 will have great impact on identifying mechanisms by which the sub-sequence factors responsible for the functioning and specificity of homeodomain proteins act. The internal dynamics of DNA are commonly speculated to be of great importance in governing specificity of protein-DNA interactions. We will employ solid-state 2H NMR methods to probe, selectively, dynamics of nucleotide bases, found within the 16 bP recognition sequence of the vnd/NK-2 homeobox, that are known to be critical to the protein-DNA interface in this complex. We will characterize dynamics of both free duplexed forms of the recognition oligonucleotide and forms bound to vnd/NK-2.

确定完整同源域的三维结构 蛋白质，以及对复式DNA的内部分子动力学的探测 专门与相关蛋白的同源物段结合 建议的。选择用于结构确定的全长蛋白是 193-遗留NKX2.8（MW = 21.9 kd），最近被发现为本地 鸡心，这是NK2班的最小成员之一 同源域蛋白。规范NK2类的内部动力学 识别DNA序列将以序列进行部分表征 复合到80分组的VND/NK-2同源蛋白质。同源域 蛋白质带有60个残留的长同源性DNA结合序列，似乎 在所有真核物种中，高度保守，进化上，包括 人类。它们是序列特异性的DNA结合因子，通常 触发基因激活的时间在胚胎发育的情况下 在普通情况下，因此在诱导细胞类型中起着作用 歧视，形态发生和生长控制。此外，染色体 替代同型杂毒的易位正常发生DNA- 转录调节器的结合段与 某些原始癌基因的致癌激活（如某些T细胞 淋巴瘤）。目前尚无完整同音域蛋白的结构。 我们建议使用高分辨率核磁共振的方法 光谱法（NMR）确定重组的溶液结构 在自由状态下生成NKX2.8。除了同型本身， NKX2.8包含另外两个已进化的子序列 在很大程度上保守。这些是12个残留的TN域， 同型蛋白N末端附近同源物的上游，23- 同型物体向C端下游的残基NK2-SD结构域。 最近在文献中报道的结果才开始提供 有关这些领域功能意义的线索。没有结构 这些域中的任何一个都存在数据。因此，发展 NKX2.8结构的详细描述将产生很大的影响 关于识别子序列因素负责的机制 对于同源域蛋白的功能和特异性，作用。这 DNA的内部动力通常被推测为非常重要 在管理蛋白质-DNA相互作用的特异性中。我们将雇用 固态2H NMR方法，有选择地探测核苷酸的动力学 基地，在VND/NK-2的16 bp识别顺序中发现 同型，已知对蛋白质-DNA界面至关重要 这个复合物。我们将表征两种自由复式形式的动态 识别寡核苷酸并与VND/NK-2结合。