Analytical ultracentrifugation

AUC samples were dialyzed into buffer containing 20 mM HEPES (pH 6.5), 100 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, and 0.02% NaN₃ and concentrated to 70 µM. Samples were loaded into cell assemblies containing Epon charcoal-filled centerpieces and quartz windows, followed by centrifugation at 45,000 rpm in an An60 Ti rotor. All sedimentation velocity experiments were collected on an Optima XL-A analytical ultracentrifuge (Beckman Coulter) at 20 °C while monitoring absorbance at 280 nm. Scans were recorded every 8 min with 0.003 cm spacing. Sedimentation profiles were analyzed with SedFit (NIH) using buffer corrections determined from SednTerp (sednterp.unh.edu; University of New Hampshire). Plots of the resultant s_20,w versus the normalized c(s) for each velocity dataset were calculated using Kaleidagraph, version 4.52 (Synergy Software).