Gel Permeation Chromatograph/Laser Light Scattering

描述(申请人提供)：生物化学的一个主要领域已经成为研究蛋白质和其他生物大分子在大型复合体中的相互作用，如转录复合体、核糖体、核糖体等。这些复合体是动态物种，随着其生化功能受到各种环境参数的调节，它们的结构会发生变化。用许多结构生物学的工具很难表征这些复合体及其构象变化，因为它们令人望而却步的大小使大多数方法不切实际。动态和静态激光光散射提供了提供这些大型复合体的粗略结构表征的可能性，同时也允许量化外部因素引起的变化。将光散射仪与尺寸排除层析系统相结合，可在光散射分析之前对络合物进行额外的分离和流体动力学表征。这可能是集成仪器的一个重要方面，因为多分散体系的光散射分析需要高度依赖于模型的复杂分析。凯斯西储大学感兴趣的第二个领域是表征参与致病淀粉样蛋白形成的蛋白质的聚集，包括阿尔茨海默病的Abeta肽(在路易小体中发现的α-突触核蛋白)和引发海绵状脑炎的普恩蛋白。在这些蛋白质的聚集过程中，低聚物形成的早期事件对最终形成不溶性淀粉样蛋白至关重要。同样，表征这些低聚物也很困难，因为它们很少是溶液中的主要形式，而是以低浓度存在于缓慢平衡或稳定的聚集体形成状态。高分辨率凝胶渗透层析系统与在线激光光散射检测器的集成将使这些淀粉样蛋白形成蛋白的不同寡聚体形式得以单独表征。同样，立即检测分离的低聚物是重要的，以避免对多分散溶液产生的贡献进行去卷积。多角度激光光散射(MALLS)检测器和准弹性光散射(QELS)检测器相结合，可以分别测定均方根半径(RG)和流体动力学半径(RH)以及洗脱液的相对分子质量。为将SEC系统与光散射组合探测器集成在一起，请提供资金，将其纳入CWRU蛋白质表征设施。

DESCRIPTION (provided by applicant): A major area of Biochemistry has become studying the interactions of proteins and other biological macromolecules in large complexes, e.g. transcription complexes, nucleosomes, ribosomes, etc. These complexes are dynamic species that modify their structure as their biochemical function is modulated by various environmental parameters. Characterizing these complexes and their conformational changes is difficult by many of the tools of structural biology as their prohibitive size makes most approaches impractical. Dynamic and static laser light scattering offer the potential to provide coarse structural characterization of these large complexes while also permitting changes induced by external factors to be quantified. Coupling the light scattering instrument to a size exclusion chromatography system permits the additional separation and hydrodynamic characterization of the complex immediately prior to the light scattering analysis. This can be an essential aspect of the integrated instrument as light scattering analysis of polydisperse systems requires complex analysis that is highly dependent on the model. A second area of concentrated interest at Case Western Reserve University is in characterizing the aggregation of proteins involved in pathogenic amyloid formation including the Abeta peptide of Alzheimer's Disease, (alpha-synuclein found in Lewy bodies, and prion proteins that trigger spongiform encephalitis. In the aggregation of these proteins, early events in the formation of oligomers are crucial to the eventual formation of insoluble amyloid. Again characterizing these oligomers is difficult because they are rarely the predominant form present in solution but exist at low concentrations in a slow equilibrium or steady state of aggregate formation. The availability of a high resolution gel permeation chromatography system integrated with an online laser light scattering detector will permit the individual characterization of the different oligomeric forms of these amyloid forming proteins. Again the immediate detection of the separated oligomer is important to avoid the necessity of deconvoluting the contributions arising from a polydisperse solution. A combination Multi-Angle Laser Light Scattering (MALLS) detector and quasi elastic light scattering (QELS) detector permit the determination of both the rms radius (RG) and the hydrodynamic radius (RH), respectively, along with the molecular weight of an eluate. Funds for the acquisition of the SEC system integrated with the combination light scattering detectors are requested for inclusion in the CWRU protein characterization facility.