Sedimentation equilibrium data fitted with a single species model indicated the presence of a species with a mass of 141,500 ± 6659 Da at infinite dilution (M 0). This corresponds with the value of s 20 ,w 0 computed (using SOMO ) for the octamer crystal structure (5.97 S) and not with that computed for the tetramer (3.62 S). The infinite dilution sedimentation coefficient ( s 20 ,w 0) of FolX, derived from the concentration dependence of s 20,w, determined by fitting the data with a non-interacting discrete species model, is 6.09 ± 0.03 S. Sedimentation velocity experiments revealed that FolX was present as a single species in solution, as evidenced by a single peak in the concentration distribution of the apparent s 20,w (c(s), Fig. In order to try to get conclusive evidence for the quaternary state of FolX, analytical ultracentrifugation was performed. Analysis of the interface between the tetrameric rings, using PDBePISA, indicates that there are 16 hydrogen bonds and 8 salt bridges connecting the two tetramers ( Supplementary Table 2), which may suggest that this interaction is significant despite the relatively small interface area. 3B), involve 7% of the solvent accessible area compared with 18% involved in stabilising the tetramer itself. The only interactions between the tetramers, in what would be a dimer of tetramers ( Fig. However, when the crystal structure is analysed by PDBePISA, it is predicted to be a tetramer. Previous studies have suggested that FolX is an octamer. Active site residues are highlighted in purple. (C) The interface between the tetrameric rings is stabilised by four hydrogen bonds (shown as dashed lines) between residues R19, R17 and E72 (shown in sticks).
(A) The tetramer ring formed by FolX dimerises to form an octamer (B). The dissociation constant was calculated using the method described by Solovyova et al. SE data were analysed using Origin and were fitted with a tetramer–octamer model with non-ideality. Scans were taken every 3 h until analysis of the scans, using WinMATCH (Jeffrey Lary, University of Connecticut, Storrs, CT, USA), indicated that equilibrium had been reached. Sedimentation equilibrium (SE) experiments were carried out with the same range of FolX concentrations using 90 μl of sample with a rotor speed of 23 krpm. The partial specific volume of FolX (0.737/0.743 g ml −1), the buffer density (1.00677/1.00499 g ml −1) and viscosity (0.0156/0.0102 P) at 4 ☌ and 20 ☌ respectively, were all calculated using the program SEDNTERP.
Data were acquired every 7 min with interference and absorbance optics and were subsequently analysed using SEDFIT. 360 μl of sample, at concentrations of FolX ranging between 0.2 and 10 mg ml −1, were loaded into double sector centrepieces. Sedimentation velocity (SV) experiments were performed at 4 ☌ at a rotor speed of 49 krpm. Analytical ultracentrifugation (AUC) was carried out in a Beckman Coulter (Palo Alto, CA) Optima XL-I analytical ultracentrifuge. Purified FolX was dialysed against 20 mM Tris pH 7.5, 150 mM NaCl and concentrated to approximately 10 mg ml −1, based on the A 280.
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