12 mm path length charcoal filled epon double sector centrepieces

Experiments were performed on a Beckman Coulter Optima XL-A analytical ultracentrifuge (Beckman-Coulter, Palo Alto, CA, USA) equipped with UV-visible absorbance detection system, using an An50Ti 8-hole rotor and 12 mm path-length charcoal-filled epon double-sector centrepieces. The experiments were carried out at a rotor speed of 48 000 rpm and 7 °C using 400 µL samples of proteins dialyzed into in PIPES buffer (20 mM PIPES, pH 7.0). Protein was at 5–20 μM and L-malic acid (stock solution made up in dialysis buffer) was added at a final concentration of 1 mM. Dialysis buffer with and without ligand were used as reference. Light at a wavelength of 234 nm was recorded in the absorbance optics mode. A least squares boundary modelling of the data was used to calculate sedimentation coefficient distributions with the size-distribution c(s) method implemented in the SEDFIT v11.71 software^{82 (link)}. The Svedberg equation allowed us to estimate the experimental molecular weight from the sedimentation and diffusion coefficients obtained. Buffer density (ρ = 1.0015 g/mL) and viscosity (η = 0.01449 Poise) at 7 °C were calculated from the buffer composition using SEDNTERP software⁸³ . This software was also used to calculate the partial specific volume (0.721 ml/g) and the molecular weight (21.5 kDa) of PA2652-LBD from its sequence.

Experiments were performed at 6 °C in a Beckman Coulter Optima XL-I analytical ultracentrifuge (Beckman-Coulter), using an An50Ti 8-hole rotor and 12 mm path-length charcoal-filled epon double-sector centrepieces. Samples were dialyzed into polybuffer in the presence or absence of Pi. Sedimentation velocity (SV) runs were carried out at 45,000 rpm using 400 μl samples. The c(s) method52 (link) implemented in the SEDFIT v14.1 software was used. Buffer density (ρ = 1.016 g/mL) and viscosity (η = 0.0177 Poise) were determined by an Anton Paar Density Meter DMA 5000 M and Microviscometer Lovis 2000 ME. The partial specific volumes were calculated from the protein sequence using SEDNTERP software53 . Sedimentation equilibrium (SE) data were acquired for 180 μl samples at speeds of 11,800, 18,100 and 31,000 rpm in the absorbance mode. The SE data were fitted using the SEDPHAT v10.55b software54 (link). Errors shown are the errors of the fit, calculated as the standard deviations using a MonteCarlo analysis with a confidence level of 0.68. This procedure is implemented in SEDPHAT54 (link).