Structure/Function Studies of Human AlphaB-Crystallin

人 AlphaB-晶状体蛋白的结构/功能研究

• 批准号: 6508346
• 负责人: Rachel E Klevit
• 金额: $ 14.39万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6508346
• 关键词: adenosine triphosphate; chimeric proteins; crystallins; electronic spectra; human tissue; mass spectrometry; molecular shape; nuclear magnetic resonance spectroscopy; protein structure function; proteolysis; stoichiometry

DESCRIPTION (provided by applicant): AlphaB-crystallin is a versatile protein. It is one of the elements essential to maintaining the transparency of the eye-lens. AlphaB-crystallin is also found up-regulated in a variety of neuro-degenerative diseases such as Alzheimer's disease and Alexander's disease. Recently, alphaB-crystallin has been shown to be a novel regulator of apoptosis. AlphaB-crystallin has been shown in vitro to be a molecular chaperone and is now classified as belonging to the family of small heat shock proteins (sHSPs) that are induced under conditions of stress. However, alphaB-crystallin (~650 kDa, ~32 subunits, 175 residues per subunit) has remained refractive to high-resolution structure studies like X-ray crystallography and solution-state NMR using conventional methods due to its the dynamic, oligomeric nature. Recent revolutionary technological and methodological advances in NMR will be used to study this challenging system using a "divide and conquer" approach. A 100-residue core domain from human alphaB forms a dimer under certain conditions of pH and temperature. The structure of the dimeric form (22.8 kDa) of the core domain will be determined using high-resolution, solution state NMR. Agents such as sodium deoxycholate are likely to dissociate the large alphaB complex into tetrameric units, analogous to acrystallin. Detailed solution state NMR studies will be performed on the alphaB-tetramer to determine the structure and orientation of the termini (which are missing in the dimeric core domain ) and thus construct a picture of "mini" alphaB. Finally, attempts will be made to obtain NMR spectra of the entire oligomeric form of alphaB using recent innovations in NMR. To understand the mechanism of chaperone-substrate interaction, NMR spectra of the core domain plus substrate will acquired using substrates like gamma-crystallin. The effects of ATP will also be determined by comparing spectra of ATP-free and ATP-bound (i) core domain and (ii) `B. Mutants and a chimeric version of alphaB-crystallin will be made to understand the functional aspects of this molecule. To supplement the information obtained from NMR, limited proteolysis mapping will be performed on the core domain/substrate and alphaB/substrate complexes to identify regions of protection. These studies will lay the foundation for more detailed investigations in the future regarding sequence determinants in the various biological roles of alphaB. If attempts to obtain spectra of the 650 kDa molecule are successful, it will open up vast avenues in NMR for studying macromolecules and their function.

描述（由申请人提供）：AlphaB-晶状体蛋白是一种多功能蛋白质。它是维持晶状体透明度所必需的元素之一。 AlphaB-晶状体蛋白在多种神经退行性疾病中也被发现上调，例如阿尔茨海默氏病和亚历山大氏病。最近，αB-晶状体蛋白已被证明是一种新型的细胞凋亡调节剂。 AlphaB-晶状体蛋白已在体外被证明是一种分子伴侣，现在被归类为属于在应激条件下诱导的小热休克蛋白 (sHSP) 家族。然而，αB-晶状体蛋白（~650 kDa，~32 个亚基，每个亚基 175 个残基）由于其动态、低聚的性质，仍然适合使用传统方法进行高分辨率结构研究，例如 X 射线晶体学和溶液态 NMR。核磁共振领域最近革命性的技术和方法进步将用于通过“分而治之”的方法来研究这个具有挑战性的系统。来自人类 alphaB 的 100 个残基核心结构域在一定的 pH 和温度条件下形成二聚体。核心结构域二聚体形式 (22.8 kDa) 的结构将使用高分辨率、溶液态 NMR 确定。脱氧胆酸钠等药物可能会将大的 αB 复合物解离成四聚体单位，类似于 acrystallin。将对 αB 四聚体进行详细的溶液态 NMR 研究，以确定末端的结构和方向（二聚体核心结构域中缺失），从而构建“迷你”αB 的图像。最后，将尝试利用核磁共振领域的最新创新来获得 alphaB 整个寡聚形式的核磁共振谱。为了了解分子伴侣-底物相互作用的机制，将使用γ-晶状体蛋白等底物获取核心结构域和底物的核磁共振谱。 ATP 的影响也将通过比较无 ATP 和 ATP 结合的 (i) 核心结构域和 (ii) `B. 的光谱来确定。突变体和 αB-晶状体蛋白的嵌合版本将被用来了解该分子的功能方面。为了补充从 NMR 获得的信息，将对核心域/底物和 alphaB/底物复合物进行有限的蛋白水解图谱，以识别保护区域。这些研究将为未来关于 alphaB 各种生物学作用的序列决定因素进行更详细的研究奠定基础。如果获得 650 kDa 分子光谱的尝试成功，将为 NMR 研究大分子及其功能开辟广阔的途径。