Sedimentation velocity experiments were performed in a Beckman Optima Xl-I analytical ultracentrifuge using conventional aluminum double-sector centerpieces and sapphire windows. Solvent density and the protein partial specific volumes were determined as described (53). Before centrifugation, dARC1 CA samples were prepared by exhaustive dialysis against the buffer blank solution, 20 mM tris-HCl (pH 8), 150 mM NaCl, and 0.5 mM TCEP (tris buffer). Samples (420 μl) and buffer blanks (426 μl) were loaded into the cells, and centrifugation was performed at 50,000 rpm and 293 K in an An50-Ti rotor. Interference data were acquired at time intervals of 180 s at varying sample concentrations (25, 50, and 100 μM). Data recorded from moving boundaries were analyzed in terms of the size distribution functions C(S) using the program Sedfit (54).

Sedimentation equilibrium experiments were performed in a Beckman Optima XL-I analytical ultracentrifuge using aluminum double-sector centerpieces in an An-50 Ti rotor. Before centrifugation, samples were dialyzed exhaustively against the buffer blank (tris buffer). Samples (150 μl) and buffer blanks (160 μl) were loaded into the cells, and after centrifugation for 30 hours, interference data were collected at 2 hourly intervals until no further change in the profiles was observed. The rotor speed was then increased, and the procedure was repeated. Data were collected on samples of different concentrations of dARC1 CA (25, 50, and 70 μM) at three speeds, and the program SEDPHAT (55) was used to determine weight-averaged molecular masses by nonlinear fitting of individual multispeed equilibrium profiles to a single-species ideal solution model. Inspection of these data revealed that the molecular mass of dARC1 CA showed no significant concentration dependency, and so, global fitting incorporating the data from multiple speeds and multiple sample concentrations was applied to extract a final weight-averaged molecular mass.

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