BIOPHYSICAL CHARACTERIZATION OF ALPHA-CRYSTALLIN

α-晶状体蛋白是脊椎动物眼透镜的主要蛋白质成分， 作为聚集体的异质群体原位存在， 约13 nm。 这些聚集体的小尺寸，加上它们的 高浓度和长期稳定性，有助于提高 图像聚焦在透镜上所需的透镜的折射率梯度 视网膜，透镜在可见波长光谱中的透明度， 和视觉中透镜功能的寿命。 然而，这种蛋白质， 与白内障的发展有关，因为 更大尺寸的可溶性颗粒和不溶性物质的群体， 主要是α-晶状体蛋白。 尽管α-晶体蛋白对 然而，在视觉功能及其在白内障发生中主要作用方面， 令人惊讶的是，关于该化合物的溶解度特性知之甚少。 亚基，聚集成颗粒的过程， 聚集状态的环境变化，或溶液的相互作用 这些粒子处于生理浓度。 这在很大程度上是由于 α-晶状体蛋白聚集体不能解离 而不伴随亚基的变性，从而防止 对蛋白质独特性质的系统探索。 亲水性 肽序列分析，结合文献分析 报告，导致了1987年由PI和一位同事制定的 α-晶状体蛋白的胶束假说，表明它的行为像 两亲分子的蛋白质形式，并在 通过疏水相互作用形成聚集体。 这一假设 使用几个独立的生物物理学测试和验证 技术，并作为实验策略设计的基础 探讨这一建议的三个具体目标： 四种α-晶状体蛋白同种型的生物物理表征; 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;以及 三种透镜混合种群的生物物理特征 低于、处于和高于生理浓度的晶体蛋白。 实现这些目标所采用的技术包括 圆二色谱法，快速液相色谱法 色谱法，流体静压应用，旋转和 振荡流变仪，渗透应力，电子显微镜，和 同步辐射散射和衍射。 制定方法， 表征单独和聚集形式的α-晶状体蛋白分子， 以及描述其在生理条件下在溶液中的相互作用 集中，是集中发展的关键先决条件。 抑制白内障发生的策略。

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.