ICAP bandshifts going well
I am using the perfect CRP binding site, ICAP, to measure both the amount of protein active in DNA binding in my protein preps and to calculate EcCRP and HiCRP’s affinities for the perfect binding site and derivatives of this site. My first affinity measurements (which are expressed as equilibrium binding constants, Kobs) indicated that I had lots of active CRP molecules, but EcCRP’s affinity for ICAP was ~100-fold less than that observed by the researchers who first developed ICAP. I am the first to work with HiCRP, so there are no precedents with which to compare HiCRP binding constants.
Initially I thought that I was skewing my Kobs measurements by using too much bait DNA in binding reactions, so I started experimenting with lower bait DNA concentrations. Changing bait DNA concentrations had no effect on Kobs measurements, which was heartening in that I have nice replicate measurements and confirms that my binding reactions are resistant to perturbations. However, this didn’t explain my low Kobs measurements.
Binding reactions are set up such that CRP is presented with a great excess of non-specific DNA; for this I use poly-dIdC, an unnatural DNA molecule that doesn’t have any CRP binding sites. Using non-specific competitor DNA ensures that non-specific DNA binding by CRP won’t contribute to the bandshifts that I am using to measure protein-DNA affinity. This is important because DNA binding proteins are attracted to DNA and so spend a lot of time interacting non-specifically with DNA; when a protein finds a specific binding site, more bonds are formed between it and the DNA so the interaction persists for a longer period. I like to think that including a great excess of non-specific DNA in bandshift reactions is the most biologically relevant approach to studying protein-DNA interactions because in a cell, the vast majority of the chromosome does not have a CRP binding site. Further, I have read and have been told that affinity constants can only be reliably measured in the presence of excess non-specific DNA.
Thus, I was surprised to discover this week as I was re-reading some ICAP papers that the ICAP gang was/is using CRP in excess over ICAP bait DNA, without any non-specific competitor! My first step was to repeat my ICAP bandshift yesterday with low CRP concentrations, but with even lower DNA concentrations (and no competitor DNA). The result is very clear: in the absence of cold competitor, the Kobs value increases ~100-fold to a value similar to previously published values. Thus, in the next few days I will delve deeper into understanding the calculation of affinity constants and will revisit those wise biochemists in the Biochem department.
No matter which approach I take with my bandshifts, I am very pleased with the quality of the data and I’m only a week away from measuring all the ICAP variants. The data will be very informative and will make a great figure for the manuscript that I think is improving by leaps and bounds.