Past exam of the biochemistry course of the University of Cambridge / 2024 / Paper 5 / Section 1 / c / Solution

The DNA-binding domain supports site occupancy, while the region for homotypic protein interaction permits much larger assemblies. With the truncated protein, the faster complex 4 appears at low protein:DNA ratios and gives way to the slower complex 3 as protein increases. A natural assignment is occupancy of one and then both recognition repeats by functional binding units. A binding unit could itself be a protein dimer; a native gel does not determine the number of polypeptide chains from mobility alone. Loss of free probe with increasing protein supports binding, and failure to shift the zero-repeat probe supports sequence preference under these conditions.

Full-length protein instead produces the very slow complex 1 already at relatively low ratios, followed by more of complex 2 at high ratios. homotypic protein interactions can make cooperative higher-order assemblies and can bridge different probe molecules. At protein excess, separate saturation of available sites can reduce productive bridging, explaining why the largest species need not increase monotonically. Complex 2 is compatible with a smaller saturated full-length assembly; assigning its exact stoichiometry requires an independent measurement. protein size also contributes to the difference between full-length and truncated complexes, but size alone does not explain the concentration-dependent redistribution into the exceptionally slow species. The experiment supports assembly dependent on protein oligomerization rather than a unique structure for every numbered band.