BIOPHYSICAL CHARACTERIZATION OF ALPHA-CRYSTALLIN

α-晶状体蛋白的生物物理特性

• 批准号: 2163709
• 负责人: Jane Koretz
• 金额: $ 23.66万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-01 至 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2163709
• 关键词: animal tissue; biophysics; chemical aggregate; circular dichroism; crystallins; electron microscopy; hydrostatic pressure; intermolecular interaction; light scattering; lipid solubility; liposomes; liquid chromatography; osmotic pressure; particle accelerators; phosphoproteins; polarimetry; protein isoforms

Alpha-crystallin, the major protein component of the vertebrate eye lens, exists in situ as a heterogeneous population of aggregates averaging about 13nm. The small size of these aggregates, combined with their very high concentration and long-term stability, contribute to the increased refractive index gradient of the lens needed for image focusing on the retina, the transparency of the lens in the visible wavelength spectrum, and the longevity of lens function in vision. The protein has, however, been implicated in the development of cataracts, as the increased population of larger-sized soluble particles and insoluble material is largely alpha-crystallin. Despite alpha-crystallin's critical importance in visual function and its major role in cataractogenesis, however, surprisingly little is known about the solubility characteristics of the subunit, the aggregation process into particles, the effects of environmental shifts on aggregation state, or solution interactions of the particles at physiological concentrations. This is due in large part to the fact that alpha-crystallin aggregates cannot be dissociated without concomitant denaturation of the subunits, preventing the systematic exploration of the protein's unique properties. Hydropathy analysis of the peptide sequence, combined with analysis of literature reports, led to the formulation in 1987 by the PI and a colleague of the micelle hypothesis for alpha-crystallin, suggesting that it behaved like the protein version of an amphipathic molecule and was stabilized in aggregate form through hydrophobic interactions. This hypothesis has been tested and validated using several independent biophysical techniques, and serves as the basis for experimental strategies designed to explore the three specific aims of this proposal: differential biophysical characterization of the four alpha-crystallin isoforms; biophysical characterization of the solution behavior of alpha-crystallin aggregates below, at and above physiological concentrations; and biophysical characterization of mixed populations of the three lens crystallins below, at, and above physiological concentrations. Techniques to be employed in the accomplishment of these aims include circular dichroism spectropolarimetry, fast performance liquid chromatography, hydrostatic pressure application, rotational and oscillatory rheometry, osmotic stress, electron microscopy, and synchrotron scattering and diffraction. Development of methods to characterize the alpha-crystallin molecule alone and in aggregate form, as well as to describe its interactions in solution at physiological concentrations, is a critical prerequisite for focused development of strategies to inhibit cataractogenesis.

α-晶体蛋白是脊椎动物眼睛晶状体的主要蛋白质成分， 以集合体的异质种群的形式存在于原位 约13 nm。这些聚集体的小尺寸，加上它们非常 高度集中和长期稳定，有助于增加 聚焦图像所需透镜的折射率梯度 视网膜，晶状体在可见光波长光谱中的透明度， 以及视力中晶状体功能的长寿。然而，这种蛋白质具有 与白内障的发展有牵连，因为 较大尺寸的可溶颗粒和不溶物质的数量是 主要是α-晶状体蛋白。尽管α-晶状体蛋白非常重要 然而，在视觉功能及其在白内障发生中的主要作用方面， 令人惊讶的是，人们对其溶解性特征知之甚少 亚单位，凝聚成粒子的过程，效果 聚合状态或解决方案相互作用的环境变化 这些颗粒在生理浓度下。这在很大程度上是由于 因为α-晶体蛋白聚集体不能解离 而不伴随亚基的变性，防止 对蛋白质独特性质的系统探索。水病 多肽序列分析与文献分析相结合 报告，导致了1987年由国际和平协会和 α-晶体蛋白的胶束假说，表明它的行为类似于 两亲性分子的蛋白质版本，并稳定在 聚集体是通过疏水作用形成的。这一假设已经 已经使用几个独立的生物物理进行了测试和验证 技术，并作为设计实验策略的基础 探讨这项建议的三个具体目标：差异化 四种α-晶体蛋白亚型的生物物理特征； α-晶状体蛋白溶液行为的生物物理表征 低于、达到和高于生理浓度的聚集体；以及 三种晶状体混合种群的生物物理特征 晶体蛋白低于、处于和高于生理浓度。 为实现这些目标而采用的技术包括 圆二色光谱旋光法，快速性能液体 层析、静水压力应用、旋转和 振荡流变仪、渗透应力、电子显微镜和 同步加速器散射和衍射。开发方法以实现 单独和以聚集形式表征α-晶状体蛋白分子， 以及描述其在生理溶液中的相互作用 集中，是集中发展的关键前提 抑制白内障发生的策略。