Happy to have been wrong about HiCRP
I conducted some very satisfying bandshifts this week, although I haven't found time to crunch and analyze the data. Thus, I will only be able to briefly describe my first impressions of the results. The first satisfaction came from confirming that my protein preps were very active in DNA binding; because preparing the proteins takes several days, I always live in fear through much of the preparation process that the final protein preps will be bunk.
The second satisfaction came from discovering that, contrary to earlier conclusions, H. influenzae CRP (HiCRP) appears to have a very high affinity for DNA, comperable to that of E. coli CRP (EcCRP). This revelation comes from testing both proteins' affinities for the theoretically optimal CRP-binding site, called "ICAP". In earlier experiments conducted in 2002 and 2005, I tested CRP affinity for a natural CRP site from the Hflu mglBAC promoter; this site was my positive control for DNA binding because it is very similar to the optimal site (ICAP). EcCRP bound the mglB site with very high affinity, but HiCRP did not. From this, I concluded that HiCRP just was not as strong a DNA binding protein. I hypothesized that HiCRP may be a weak DNA binding protein because it does not form very stable dimers (only homodimeric CRP molecules bind DNA). I think that my ICAP results may offer some support for this theory. Although HiCRP and EcCRP demonstrated a similar affinity for ICAP when they were in the 10-500 nM concentration range, my impression was that HiCRP binding was not detectable below 10 nM, whereas EcCRP binding could be detected down to 0.5 nM. In my thesis I lamented that I did not know of a good assay to detect dimerization of CRP molecules at the low concentrations where dimerization would be an important contributer to CRP's ability to bind DNA. However, bandshift assays are very sensitive to inter-molecular interactions at very low protein and DNA concentrations, so perhaps I will be able to glean some more informative data from my bandshifts than I had initially intended. This will require that I get a better handle on the calculations used and underlying assumptions made when measuring protein-DNA binding kinetics.