NMR STUDIES OF THE C-TERMINAL DOMAIN OF TENASCIN

这项研究计划描述了核磁共振(NMR) 26kD C末端“纤维蛋白原样区”的结构测定 Tenascin的蛋白质。Tenascin是一种细胞外基质蛋白 在发育早期表达，并在成体组织中重新表达 在愈合过程中，并与肿瘤转移相关。许多 的已知活性定位于C-末端结构域 它显示细胞和肝素结合活性。另外， Tenascin的C-末端结构域具有显著的序列同源性 其他非常有趣的蛋白质，没有一个是结构上的 下定决心。重组C-末端结构域的完全归属 将使用最先进的异核材料制造 (1H，13C，15N)3D和4D核磁共振方法。NOE、J偶联与氢键 限制条件将使用各种异核核磁共振来获得 技巧。这些约束将与模拟一起使用 用退火法和约束分子动力学生成结构 模特们。NOE强度的反算将用于验证和 改进这些核磁共振结构。松弛参数t1、t2和 异核NOE将被测量并适用于各种弛豫模型 提取分子内运动参数。这一信息将是 用于定义与细胞和肝素结合有关的可能区域 网站。Tenascin的C-末端的详细原子分辨结构 结构域在定位特定氨基酸方面应该是非常有价值的 对结合活动和指导未来的诱变和 Tenascin在肿瘤转移和转移中作用的生物学研究 组织发育。

This research proposal describes the nuclear magnetic resonance (NMR) structure determination of the 26 kD C-terminal "fibrinogen-like' domain of the protein tenascin. Tenascin is an extracellular matrix protein expressed during early development and is reexpressed in adults tissues during healing processes and in association with tumor metastasis. Many of tenascin's known activities are localized to the C-terminal domain which displays cell and heparin binding activities. Additionally, tenascin's C-terminal domain shares significant sequence homology to other highly interesting proteins, none of which have been structurally determined. The complete assignment of the recombinant C-terminal domain of tenascin will be made using state-of-the-art heteronuclear (1H,13C,15N) 3D and 4D NMR methods. NOE, J-coupling and hydrogen bonding constraints will be obtained using a variety of heteronuclear NMR techniques. These constraints will be used in conjunction with simulated annealing and constrained molecular dynamics to generate structural models. The backcalculation of NOE intensities will be used to verify and improve these NMR structures. The relaxation parameters T1,T2 and the heteronuclear NOE will be measured and fit to various relaxation models to extract intramolecular motional parameters. This information will be used to define possible regions implicated in cell and heparin binding sites. The detailed atomic resolution structure of tenascin's C-terminal domain should be invaluable in localizing the specific amino-acids critical to binding activities and in guiding future mutagenesis and biological investigations into tenascin role in tumor metastasis and tissue development.