Analytical Ultracentrifugation for Studying the Oligomeric State and Protein-Protein Interactions of Membrane...

Analytical ultracentrifugation is still the gold-standard technique for detecting and characterizing the interactions between particles in solution, including membrane transporter proteins. However, because membrane transporter proteins require detergents (and sometimes phospholipids) to solubilize the protein in the aqueous phase, analytical ultracentrifugation is not as simple as for soluble proteins. Here, we present and develop our protocol for carrying out sedimentation velocity-type analytical ultracentrifugation experiments to help understand the mechanism of multi-component tripartite ATP-independent (TRAP) transporter proteins, which are essential for nutrient uptake in bacteria. We describe the expression and purification of TRAP transporters in detergent and exchange them into alternative membrane mimetics, including amphipol and nanodiscs, and we outline the methodology to determine TRAP transporter stoichiometry, define the average number of detergent molecules that are bound to the protein, and perform in-solution studies in native-like membrane environments free from detergents. These methodologies can be applied to other membrane transporters and complexes to answer fundamental questions related to size, shape, stoichiometry, and interactions with other proteins in solution.