Latest ICAP results
I have now quantified EcCRP and HiCRP affinity for ICAP and four designer variants, and am currently replicating the experiments. The data looks good and supports my hypotheses, but some additional interesting features of CRP-DNA interactions have revealed themselves.
For example, CRP binding causes DNA to assume a very sharp bend of around 90º, which is achieved through two major kinks near the centre of the DNA site (each ~40º) and lesser kinks at each edge of the binding site (each ~10º). Two of the ICAP variants were designed (in part) to address the importance of the small secondary kinks for HiCRP affinity. As I predicted, HiCRP appears to need favourable interactions with a longer stretch DNA than does EcCRP, possibly to stabilize kinking, but what I didn’t expect is that I can readily detect variations in the degree to which DNA is bent by CRP. The second surprise is that when multiple CRP molecules bind to bait DNA at high protein concentrations in bandshift reactions, HiCRP appears to bind in a stepwise fashion: first one protein and then two (possibly in a cooperative manner). EcCRP, on the other hand, goes very quickly from having only one protein bound to a stage were more than two proteins bind to the same piece of DNA, seemingly in a more haphazard fashion. I suspect this is consistent with my model in which HiCRP is highly selective for DNA sites, whereas EcCRP is much less choosy and will bind all sorts of less favourable sites when the good ones are saturated.
These are interesting results, but they require much more thinking before I make good sense of them. Also, they beg for more experiments and my plate is looking pretty full considering the number of different DNA species (ie. different natural promoters) I still want to test in "simple" affinity experiments.