1. Assembly of MtGimα and MtGimβ into a hexameric complex with high α‐helical content.
Assembly of MtGimα and MtGimβ into a hexameric complex with high α‐helical content. (A) Size exclusion chromatography (on a Superdex 200 column) of refolded MtGimβ, MtGimα, and mixtures of MtGimβ and MtGimα in 1:1, 2:1 and 3:1 molar ratios. The left panel shows fractions analysed by SDS–PAGE (16%) and Coomassie Blue staining, and the right panel shows the elution profiles (A280) of the samples containing different MtGimβ to MtGimα ratios. MtGimC represents the complex between MtGimα and MtGimβ. (B) Cross‐linking reactions of MtGimα and MtGimβ (each 20 μM) using 1 mM BS3 for 1 h, analysed by SDS–PAGE and Coomassie Blue staining. (C) Sedimentation equilibrium analysis of purified MtGimC performed at three different protein concentrations (see Materials and methods). RM was 5.9, 6.4 and 6.9 cm for the high, intermediate and low concentrations, respectively. The data obtained fit well to a single ideal species with a molecular mass of 83.7 kDa. (D) Circular dichroism measurements of MtGimα, MtGimβ and MtGimC in 20 mM NaPO4 pH 8.0 at 20°C. All of the samples examined have a high α‐helical structure content; calculations suggest that MtGimC has an α‐helical content of ∼85%.
Michel R. Leroux et al. EMBO J. 1999;18:
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