Light Scattering Techniques to Assess Self-Assembly and Hydrodynamics of Membrane Trafficking Proteins.

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.