Additional file 3.
Mutants of the secondary patch reduce oligomerisation. Mutagenesis of surface residues. (a) Oligomerisation of partially purified (see Methods) 80 μM WT or mutant Irga6 proteins was monitored by light scattering in the presence of 10 mM GTP at 37°C. Left panel: positive (WT) and negative (K196D) control (Figure 2a). Right panel: investigated mutants. Five mutants R31E-K32E, K169E, K176E, R210E and K246E inhibited the oligomerisation of Irga6, whereas many others had no significant effect. The mutants were fully purified. (b) Oligomerisation of 80 μM WT or mutant Irga6 proteins was monitored by light scattering in the presence of 10 mM GTP at 37°C. (c) Hydrolysis of 10 mM GTP (with traces α32P-GTP) was measured in the presence of 80 μM WT or mutant Irga6 proteins at 37°C. Samples were assayed by TLC and autoradiography.
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Pawlowski et al. BMC Biology 2011 9:7 doi:10.1186/1741-7007-9-7