Mrozowich Tyler, McLennan Steffane, Overduin Michael, Patel Trushar R
Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge.
Department of Biochemistry, University of Alberta.
J Vis Exp. 2018 Nov 5(141). doi: 10.3791/58538.
Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin γ-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.
涉及具有多个球状结构域的蛋白质之间的蛋白质-蛋白质相互作用，在确定此类复合物如何形成以及结构域如何定向/定位方面存在技术挑战。在此，描述了一种有可能通过从头建模来阐明多组分系统中哪些特定结构域介导相互作用的方案。提供了一种计算大分子及其组装体溶液结构的方法，该方法涉及以混合方法将来自小角X射线散射（SAXS）、色谱和原子分辨率结构的数据整合在一起。一个具体的例子是全长巢蛋白-1复合物，它组装细胞外基质蛋白并形成一个延伸的、弯曲的纳米结构。其一个球状结构域附着于层粘连蛋白γ-1，后者构成基底膜。这为确定柔性多结构域蛋白质复合物的精确结构提供了基础，并且通过同步辐射源与自动化机器人技术和尺寸排阻色谱系统相结合得以实现。这种组合允许进行快速分析，即在SAXS数据收集之前分离多种寡聚状态。该分析可得出关于回转半径、颗粒尺寸、分子形状和结构域间配对的信息。还给出了通过拟合组成蛋白质的高分辨率结构来生成复合物三维模型的方案。
