Gel Permeation Chromatograph/Laser Light Scattering

描述（申请人提供）：生物化学的一个主要领域是研究蛋白质和其他生物大分子在大型复合物中的相互作用，如转录复合物、核小体、核糖体等。这些复合物是动态的物种，当它们的生化功能被各种环境参数调节时，会改变它们的结构。结构生物学的许多工具很难描述这些复合物及其构象变化的特征，因为它们的令人望而却步的大小使得大多数方法不切实际。动态和静态激光散射提供了提供这些大型配合物的粗略结构表征的潜力，同时也允许由外部因素引起的变化被量化。将光散射仪器与尺寸排除色谱系统耦合，可以在光散射分析之前立即对配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，因为多分散系统的光散射分析需要高度依赖于模型的复杂分析。凯斯西储大学关注的第二个领域是表征与致病性淀粉样蛋白形成有关的蛋白质聚集，包括阿尔茨海默病的β肽、路易小体中发现的α -突触核蛋白和引发海绵状脑炎的朊病毒蛋白。在这些蛋白质的聚集过程中，低聚物形成的早期事件对不溶性淀粉样蛋白的最终形成至关重要。再次，表征这些低聚物是困难的，因为它们很少是溶液中的主要形式，而是在低浓度下以缓慢的平衡或稳定的聚集形成状态存在。结合在线激光散射检测器的高分辨率凝胶渗透色谱系统的可用性将允许这些淀粉样蛋白形成蛋白的不同低聚形式的个体表征。再次，立即检测分离的低聚物对于避免对多分散溶液产生的贡献进行反卷积的必要性是重要的。结合多角度激光光散射（MALLS）检测器和准弹性光散射（QELS）检测器，可以分别测定洗脱液的均数半径（RG）和流体动力半径（RH），以及洗脱液的分子量。申请资金用于购买SEC系统和组合光散射检测器，用于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.