NMR STUDIES OF THE C-TERMINAL DOMAIN OF TENASCIN

该研究方案描述了核磁共振（NMR） 26kD纤维蛋白原样结构域的结构测定 蛋白质腱生蛋白的一部分肌腱蛋白是一种细胞外基质蛋白 在早期发育过程中表达，并在成年组织中重新表达 在愈合过程中并与肿瘤转移有关。许多 腱生蛋白的已知活性定位于C-末端结构域 其显示细胞和肝素结合活性。此外，本发明还 腱生蛋白的C-末端结构域与 其他非常有趣的蛋白质，没有一个在结构上是 测定重组体C-末端结构域的完整分配 将使用最先进的异源蛋白 (1H、13 C、15 N）3D和4D NMR方法。NOE、J-偶联和氢键 约束将获得使用各种异构NMR 技术.这些约束将与模拟的 退火和约束分子动力学以产生结构 模型NOE强度的反算将用于验证和 改进这些NMR结构。弛豫参数T1、T2和 将测量异方差NOE并将其拟合到各种松弛模型中 提取分子内运动参数。此信息将 用于定义涉及细胞和肝素结合的可能区域 网站.腱生蛋白C-末端的原子级结构 域应该是非常宝贵的定位特定的氨基酸 对结合活性和指导未来诱变至关重要， 生腱蛋白在肿瘤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.