哺乳动物dynactin的CAP-Gly域的固态和溶液NMR研究及其与微管的相互作用
Shangjin Sun1, Amanda Siglin, John C Williams
1Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Journal of the American Chemical Society
|July 8, 2009
概括
固态NMR成功地表征了CAP-Gly/微管复合体,揭示了蛋白质结合时的结构变化. 这种技术对于在原子层面研究微管蛋白相互作用是有价值的.
科学领域:
- 生物物理学的生物物理.
- 结构生物学 结构生物学
- 生物化学 生物化学
背景情况:
- 微管 (MTs) 和微管结合蛋白 (MTBPs) 对于细胞功能如运输和分裂至关重要.
- 获取有关MT/MTBP相互作用的原子级结构和动态信息是具有挑战性的,因为MT不可溶性和缺乏顺序.
研究的目的:
- 为了研究哺乳动物dynactin的CAP-Gly域与paclitaxel稳定微管之间的相互作用.
- 评估神奇角度旋转固态核磁共振对这些复合体的特征的有用性.
主要方法:
- 结合魔术角度旋转 (MAS) 固态和溶液NMR光谱学.
- 使用2D和3D同核和异核相关性谱 (例如,DARR,HSQC).
- 分析了自由和绑定的CAP-Gly的共振分配和二次结构.
主要成果:
- 通过使用固态NMR,在自由状态和微管结合状态中实现了CAP-Gly的残留特异性共振赋值.
- 在CAP-Gly与微管结合时,在固态光谱中观察到显著的化学转移干扰,表明了形状变化.
- 溶液NMR (HSQC) 显示在结合时峰值扩大,阻碍了结构分析.
结论:
- 固态NMR是一种可行的技术,用于CAP-Gly/微管组件的详细结构特征.
- MAS固态NMR可以克服溶液NMR在研究不溶性MT/蛋白质复合物的局限性.
- 这种方法使我们能够了解基本细胞机械的动态和结构.
相关概念视频
01:17Studying the Cytoskeleton
The cytoskeletal architecture can be studied using different microscopic and biochemical techniques. Electron microscopy was instrumental in discovering the cytoskeletal architecture around the 1960s, which allowed obtaining structural information at a high-resolution level. However, the sample preparation procedure often limits this ability in biological samples. Several protocols have been developed over the years to optimize sample preparation. In one of the protocols known as rotary...
01:32Pinching-off of Coated Vesicles
Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, membrane-bending proteins containing crescent-shaped BAR domains bind to the lipid heads in the bilayer and distort the membrane to form a protein-coated vesicle bud. Adaptors proteins such as AP2 for clathrin-coated vesicles can nucleate on the deformed membrane. Finally, coat proteins such as clathrin or COPI and COPII assemble into a coat forming...
01:18Microtubules
Microtubules are the thickest cytoskeletal filaments with a diameter of 25 nm. In prokaryotic organisms, microtubules are commonly found in locomotory appendages like cilia and flagella. In eukaryotic cells, microtubules form specialized extensions for moving fluid over the surface, like those found in cells lining the intestine.
Microtubules have two structurally similar globular protein subunits: α and β tubulins. In the cytosol, the α and β tubulins form a heterodimer. These αβ-heterodimers...
Microtubules have two structurally similar globular protein subunits: α and β tubulins. In the cytosol, the α and β tubulins form a heterodimer. These αβ-heterodimers...
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...