Light Scattering Techniques to Assess Self-Assembly and Hydrodynamics of Membrane Trafficking Proteins.
Light Scattering Techniques to Assess Self-Assembly and Hydrodynamics of Membrane Trafficking Proteins.
复制标题
光散射技术评估膜运输蛋白的自组装和流体动力学。
DOI：
10.1007/978-1-0716-2209-4_18
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Ramachandran,Rajesh
中科院分区：
文献类型：
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作者：
Ford,MarijnGJ;Ramachandran,Rajesh
光散射法可以从第一性原理确定蛋白质的摩尔质量和流体动力学半径。因此，它们对任何蛋白质的生物物理特性都特别有用。摩尔质量和流体动力学半径的测定可以用来证明目标蛋白质是多聚体。在内膜系统中，蛋白质的可逆和可调控的组装和多聚化对于构建囊泡萌发所需的涂层、膜重塑机器的功能、分裂和融合以及运输中间产物的组装和分解至关重要。因此，光散射方法极大地促进了对基本贩运过程的了解。在这里，我们描述了表达和纯化重组真菌SNX-bar Mvp1的方法，这是一种内吞体内逆行运输所需的膜重塑蛋白。以Mvp1为例，给出了用多角度静态光散射法和动态光散射法分别确定其摩尔质量和流体动力学半径的方法。这些方法可以直接应用于其他膜转运蛋白的研究，产生丰富的生物物理和生化信息。
Light scattering methods permit the determination of molar mass and hydrodynamic radius for a protein from first principles. They are, therefore, particularly useful for the biophysical characterization of any protein. Molar mass and hydrodynamic radius determinations may be used to demonstrate that the protein of interest multimerizes. In the endomembrane system, reversible and regulated assembly and multimerization of proteins is critical for building coats required for vesicle budding, for the function of membrane remodeling machines, for fission and fusion and for assembling and disassembling trafficking intermediates. Light scattering methods have therefore significantly contributed to the understanding of the underlying trafficking processes. Herein, we describe methods to express and purify the recombinant fungal SNX-BAR Mvp1, a membrane remodeling protein required for retrograde trafficking at the endosome. Using Mvp1 as an example, we provide protocols for determining its molar mass and hydrodynamic radius by multiangle static light scattering and dynamic light scattering, respectively. These methods can be applied directly to the study of other membrane trafficking proteins, yielding a wealth of biophysical and biochemical information.