BIOPHYSICAL CHARACTERIZATION OF ALPHA-CRYSTALLIN

α-晶状体蛋白的生物物理特性

• 批准号: 2163710
• 负责人: Jane Koretz
• 金额: $ 18.11万
• 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/2163710
• 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-晶状体蛋白，脊椎动物眼睛晶状体的主要蛋白质成分， 作为聚合平均的异质群体原位存在 约13纳米。 这些聚集体的尺寸很小，加上它们的非常 高浓度和长期稳定性，有助于增加 图像聚焦在透镜上所需的折射率梯度 视网膜，晶状体在可见波长光谱中的透明度， 以及晶状体视觉功能的寿命。 然而，该蛋白质具有 与白内障的发展有关，随着白内障的增加 较大尺寸的可溶性颗粒和不溶性物质的总体是 主要是α-晶状体蛋白。 尽管 α-晶状体蛋白至关重要 然而，视觉功能及其在白内障发生中的主要作用 令人惊讶的是，人们对这种物质的溶解度特性知之甚少。 亚基，聚集成颗粒的过程，影响 聚集状态的环境变化，或溶液相互作用 生理浓度的颗粒。 这很大程度上是由于 事实上，α-晶状体蛋白聚集体无法解离 不伴随亚基变性，防止 系统地探索蛋白质的独特性质。 水疗 肽序列分析，结合文献分析 报告，导致 PI 和一位同事于 1987 年制定 α-晶状体蛋白的胶束假说，表明它的行为类似于 两亲分子的蛋白质版本并稳定在 通过疏水相互作用形成聚集体。 这个假设有 使用多个独立的生物物理学进行了测试和验证 技术，并作为设计实验策略的基础 探讨该提案的三个具体目标：差异化 四种 α-晶状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、等于和高于生理浓度的聚集体；和 三个晶状体混合群体的生物物理特征 低于、等于和高于生理浓度的晶状体蛋白。 实现这些目标所采用的技术包括 圆二色性光谱偏振法，快速性能液体 色谱法、静水压应用、旋转和 振荡流变测定法、渗透应力、电子显微镜和 同步加速器散射和衍射。 开发方法 单独和聚集形式表征 α-晶状体蛋白分子， 以及描述其在生理溶液中的相互作用 集中精力，是重点发展的重要前提 抑制白内障发生的策略。