Abstract
Determination of the physical parameters underlying protein-DNA interactions is crucial for understanding the regulation of gene expression. In particular, knowledge of the stoichiometry of the complexes is a pre-requisite to determining their energetics and functional molecular mechanisms. However, the experimental determination of protein-DNA complex stoichiometries remains challenging. We have used fluorescence cross-correlation spectroscopy (FCCS) to investigate the interactions of the control catabolite protein of gluconeogenic genes (CcpN), a key metabolic regulator in Gram positive bacteria, with two oligonucleotides derived from its target operator sequences, gapB and pckA. According to our FCCS experiments, the stoichiometry of binding is two-fold larger for the pckA target than for gapB. Correcting the FCCS data for protein self-association indicated that CcpN forms dimeric complexes on the gapB target and tetrameric complexes on the pckA target. Analytical ultracentrifugation (AUC) coupled with fluorescence anisotropy and hydrodynamic modeling allowed for unambiguous confirmation of this result. The use of multiple complementary techniques to characterize these complexes should be employed wherever possible. However, there are cases in which AUC is precluded, either due to protein stability, solubility or availability, or more obviously, when the studies are carried out in live cells. If information concerning the self association of the protein is available, FCCS can be used for the direct and simultaneous determination of the affinity, cooperativity and stoichiometry of protein-DNA complexes in a concentration range and conditions relevant to the regulation of these interactions.