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To clarify the composition of the complex, the oligomerization states of EIIAgnt, EIIBgnt and the complex were investigated by AUC. With equilibrium AUC experiments, molecular masses of 32, 22 and 69 kDa were measured for EIIAgnt, EIIBgnt and the EIIAgnt:EIIBgnt complex, respectively, indicating that EIIAgnt is present as a dimer (Fig. 2), EIIBgnt as a GSK690693 and the complex as a heterotetramer with a 2:2 ratio of EIIAgnt to EIIBgnt (Table 1). All data could be fit with single species models, not suggesting the presence of any other oligomeric states or equilibria. From the AUC data, we further conclude that EIIAgnt and EIIBgnt interact in a high affinity complex, consistent with the high association constant measured by ITC (see above). Additional sedimentation velocity AUC experiments allowed the determination of sedimentation coefficients for EIIA, EIIB and the complex, confirming the oligomeric states observed by equilibrium centrifugation measurements (Table 1 and Fig. 2).
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