FIG. 6.

Assembly of the cyclic cellulosome. (A) Electrophoretic mobility of components and assembled complexes. Lane 1, M-C-48t alone; lane 2, M-T-9f alone; lane 3, M-F-48t alone; lane 4, M-T-9c alone; lane 5, complex 1 (M-C-48t plus M-T-9f); lane 6, complex 2 (M-F-48t plus M-T-9C); lane 7, complex 1 plus complex 2 (cyclic cellulosome). In lanes 1 to 6, equimolar concentrations (10 μM) of the indicated proteins were used. In lane 7, complexes 1 and 2 were mixed at a final concentration of 5 μM. (B) Gel filtration analysis of the assembled complexes. Injected proteins (100 μl) and the corresponding lanes in panel A are indicated on each chromatogram. mAU refers to milli absorbance units at 280 nm. Vertical lines indicate the positions of molecular mass markers: blue dextran or void volume (>2 MDa), ferritin (440 kDa), aldolase (158 kDa), and bovine serum albumin (67 kDa). For each chromatogram, the indicated proteins were at a concentration of 10 μM, except for cyclic cellulosome, where 5 μM concentrations of complexes 1 and 2 were used. The peak observed at 20 ml in all chromatograms corresponds to the maleic acid contained in the sample buffer. Note that the formation of complex 2 generates a major and minor band on nondenaturing PAGE (panel A, lane 6), but a single peak at the expected mass on gel filtration (panel B). Since the mobilities of the bands differ from those observed for free components (panel A, lanes 3 and 4), it is assumed that the two bands correspond to two different conformations of complex 2.