Xenbase Image ID: 127148

Figure 4. Gel filtration studies and sedimentation velocity ultracentrifugation analysis of TTL binding to tubulin. (a–e) Complex formation between TTL or TTL mutants and tubulin in various nucleotide conditions monitored by gel filtration chromatography. Binding is monitored by the disappearance of the slower mobility peak (corresponding to uncomplexed TTL) when TTL or TTL mutants are incubated with excess tubulin (68 µM tubulin and 34 µM TTL or TTL mutant). The gels correspond to peak fractions indicated in green. (f) Sedimentation coefficient distributions (c(s)) obtained for 10 µM TTL and 4.8 µM tubulin (g) c(s) for TTL–tubulin complexes at varying concentrations. All complexes are at 1:1 stoichiometry, except that shown in the cyan trace, which denotes a 1:4 tubulin:TTL ratio. The sedimentation coefficient distributions obtained with mixtures of 1:1 tubulin:TTL ratio at increasing total concentrations show two peaks, a small s value peak corresponding to free TTL, with the area of the peak decreasing with increasing protein concentration, and the high s value peak representing the position of the TTL–tubulin complex reaction boundary. At lower concentrations, the position of the higher s value peak corresponds to that of the tubulin dimer alone and increases in s value with increasing concentration. By analyzing the shift in position of the complex peak with increasing protein concentration the affinity constant of TTL for tubulin was determined to be ~1 µM (method described in56; Methods). When TTL is in excess so that all tubulin is in complex with TTL, a narrow symmetrical boundary of TTL–tubulin is observed. Image published in: Szyk A et al. (2011) Image downloaded from an Open Access article in PubMed Central. Image reproduced on Xenbase with permission of the publisher and the copyright holder. Larger Image Printer Friendly View

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