DdBrk1 forms stable trimers in solution.

<p>Analytical ultracentrifugation experiments of 43 µM DdBrk1 in PBS at a detection wavelength of 280 nm. (A) Sedimentation equilibrium gradients were measured at rotor speeds of 18,000 rpm and 26,000 rpm at 10°C. Global fitting of the data with a model of a single species using the program BPCfit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021327#pone.0021327-Witte1" target="_blank">[46]</a> yielded a molar mass of 24.7 (±2) kg/mol (solid lines) indicating that the protein forms trimers in solution. (B) Sedimentation coefficient distribution as obtained from a sedimentation velocity experiment at 20°C using the program SEDFIT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021327#pone.0021327-Schuck1" target="_blank">[48]</a>. DdBrk1 sediments as a single species with a sedimentation coefficient s_20,W = 2.1 S. Experiments performed in a concentration rage of 15 µM to 340 µM DdBrk1 gave no indication of aggregation or dissociation of the DdBrk1 trimers, as indicated by a slight decrease of the sedimentation coefficient with increasing protein concentration (data not shown). The inset shows a Coomassie stained 16%-Tris/Tricin SDS-PAGE of the DdBrk1 sample used in these experiments.