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

四位申请人（Stetefeld博士、McKenna博士、Khajehpour博士和O 'Neil博士）实验室的研究项目集中于研究各种有趣的生物系统，包括具有催化活性的膜蛋白和膜蛋白通道、叶绿素生物合成酶、来自美洲驼的单克隆抗体、病毒核酸识别蛋白、调节性细菌激酶和转录激活剂。连接每个研究小组的共同特征是特定蛋白质样品及其潜在配体的结构和功能表征，以研究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本提案中包括的研究小组能够确定蛋白质-配体复合物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其生化功能和机制。作为这一战略的一个组成部分，光散射方法结合尺寸排阻色谱法，允许在溶液中的蛋白质和相关的蛋白质-配体复合物的分子特征的绝对测定已成为蛋白质结构/功能研究实验室的中心特征。要求的分子排阻色谱-多角度光散射仪器（SEC-MALS）提供了一种有价值的工具，（i）探索在尺寸分离的样品的每个点处的绝对分子量信息，（ii）鉴定蛋白质和相关蛋白质-配体复合物及其缔合状态，（iii）检测高阶聚集体和蛋白质缀合物，和（iv）检测缀合物的分子量以快速和明确地确定化学计量。当与我们研究小组现有的基础设施相结合时，SEC-MALS提供了一种强大的手段来分析生物分子相互作用，并研究蛋白质结合和蛋白质稳定性的分子细节。马尼托巴省目前没有SEC-MALS仪器。因此，显然迫切需要获得这一关键的基础设施。

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.