(A) Trypsin digestion of pre-assembled WT and mutant small Tim complexes at RT for 30 min. The samples were analysed using 16% Tris/Tricine SDS/PAGE and Coomassie staining. (B) As in (a) but 2 μg/ml trypsin was used for the indicated times and the samples were analysed by Western blotting with antibodies against Tim9 and Tim10 respectively (C) Thermal denaturation of the pre-assembled WT and mutant small Tim complexes measured by CD at 222 nm.