Efficient Protein and Protein-Ligand complex characterization via SEC-MALS

四位申请者(Stetefeld博士、McKenna博士、Khajehour博士和O‘Neil博士)的实验室研究计划集中于研究各种有趣的生物系统，包括具有催化活性的完整膜蛋白和完整的膜蛋白通道、叶绿素生物合成酶、骆驼单抗、病毒核酸识别蛋白、调节细菌激酶和转录激活剂。将每个研究小组联系在一起的共同特征是特定蛋白质样本及其潜在配体的结构和功能表征，以调查它们在调节感兴趣的生物系统中的作用。结构生物学领域的快速发展使这一提议中包括的研究小组能够确定蛋白质-配体复合体的高分辨率三维图像，并开始根据决定相互作用的关键分子特征来表征它们的生化功能和机制。作为这一策略的一个组成部分，光散射方法与尺寸排除层析相结合，允许绝对确定蛋白质在溶液中的分子特征和相关的蛋白质-配体复合体，已成为蛋白质结构/功能研究实验室的中心特征。所要求的尺寸排除色谱-多角度光散射仪(SEC-MALS)提供了一个有价值的工具：(I)探索大小分离样品每个点的绝对分子质量信息，(Ii)识别蛋白质和相关的蛋白质-配体复合体及其结合状态，(Iii)检测高阶聚集体和蛋白质结合物，以及(Iv)检测结合物的分子质量，以快速和明确地确定化学计量比。与我们的研究小组现有的基础设施相结合，SEC-MALS为分析生物分子相互作用和研究蛋白质结合和蛋白质稳定性的分子细节提供了强大的手段。马尼托巴省目前没有SEC-MALS仪器。因此，购买这一至关重要的基础设施显然是迫切需要的。

Research programs in the laboratories of the four applicants (Drs. Stetefeld, McKenna, Khajehpour and O'Neil) are focused on investigating a variety of interesting biological systems including integral membrane proteins with catalytic activities and integral membrane protein channels, a chlorophyll biosynthesis enzyme, monoclonal antibodies from llama, viral nucleic acid recognition proteins, a regulatory bacterial kinase and a transcription activator. The common feature that links each of these research groups is the structural and functional characterization of specific protein samples and their potential ligands to investigate their role in regulating the biological systems of interest. Rapid advances in the field of structural biology have allowed the research groups included in this proposal to determine high-resolution three-dimensional images of protein-ligand complexes, and begin to characterize their biochemical function and mechanism in terms of key molecular features that dictate the interaction. As an integral component of this strategy, light scattering approaches in combination with size exclusion chromatography that allow the absolute determination of molecular features of a protein in solution and the relevant protein-ligand complex have emerged as a central feature of protein structure/function research laboratories. The requested Size Exclusion Chromatography-Multiple Angle Light Scattering instrument (SEC-MALS) provides a valuable tool (i) to explore the absolute molecular mass information at every point of the size-separated sample, (ii) to identify protein and relevant protein-ligand complexes and their association state, (iii) to detect higher-order aggregates and protein conjugates, and (iv) to detect the molecular mass of the conjugates to rapidly and unambiguously determine stoichiometry. When combined with the already existing infrastructure accessible to our research groups, SEC-MALS provides a powerful means to analyze biomolecular interactions and to study molecular details of protein binding and protein stability. No SEC-MALS instrument is currently available in the Province of Manitoba. Therefore there is a clear and urgent need for the acquisition of this crucial piece of infrastructure.