HOW SITE DIRECTED MUTANTS AFFECT HBS FIBER STRUCTURE

定点突变体如何影响 HBS 纤维结构

• 批准号: 6606070
• 负责人: ROBERT JOSEPHS
• 金额: $ 31.37万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606070
• 关键词: cell component structure /function; cell morphology; electron microscopy; hemoglobin Ss; human tissue; intracellular; molecular assembly /self assembly; polymerization; protein structure function; site directed mutagenesis; video microscopy

DESCRIPTION (provided by applicant): The intracellular polymerization of sickle cell hemoglobin to form rod-like fibers causes sickle cell anemia. Cells containing fibers become distorted and rigid and occlude small capillaries causing tissue destruction. The structure of the fibers is of interest since this information could provide a rational basis for therapies aimed at inhibiting fiber formation. In addition, understanding the structural basis of HbS polymerization offers insights relevant to other systems involving macromolecular assemblies. We have succeeded in carrying out reconstructions of electron micrographs of frozen-hydrated HbS fibers. By averaging 10 fibers into the reconstruction we have improved the resolution of the reconstruction from 32A (previously published) to 16A radially and 8A axially. The improvement in resolution is caused by increasing the signal to noise ratio. We believe that the frozen-hydrated fibers are sufficiently well preserved that we could attain a resolution of at least 4 -6A by averaging several hundred particles into the reconstruction. A second series of experiments involves synthesis of double mutants (the HbS site and one other site) to explore how changes in individual amino acids effect the fiber properties. Site directed mutants at intermolecular contacts provide a means of critically testing current molecular models of the fiber structure by their effect on polymerization. We propose to study the structure of fibers containing the site directed mutants by electron microscopy and polymerization kinetics by video enhanced light microscopy.

描述（由申请人提供）： 镰状细胞血红蛋白的细胞内聚合形成棒状纤维会导致镰状细胞 贫血。包含纤维的细胞变形，刚性并阻塞小毛细血管 导致组织破坏。纤维的结构很有趣，因为此信息可以 为旨在抑制纤维形成的疗法提供合理的基础。此外， 了解HBS聚合的结构基础提供了与涉及大分子组件的其他系统相关的见解。 我们已经成功地重建了对冷冻水合的HBS纤维的电子显微照片。通过将10个纤维纳入重建，我们将重建的分辨率从32a（以前发表）提高到了径向的16a，轴向上的8a。分辨率的改善是由增加信号与噪声比的增加引起的。我们认为，冷冻水合的纤维足够保存得很好，我们可以通过将几百个颗粒的平均颗粒平均到重建中达到至少4 -6a的分辨率。 第二系列实验涉及合成双突变体（HBS位点和另一个位点），以探讨单个氨基酸的变化如何影响纤维性质。位于分子间接触的位点突变体通过对聚合的影响，为纤维结构的电流分子模型提供了一种方法。我们建议通过电子显微镜和聚合动力学来研究包含位点的纤维的结构，并通过视频增强的光学显微镜进行。