Tuesday, September 18, 2007

Measuring how tightly CRP binds to a perfect CRP-binding site

It is important to know how tightly a transcription factor (or other protein regulator) binds to a specific target DNA site because this will dictate how long the transcription factor occupies that site. In the case of target sites in gene promoters, excessive affinity for DNA results in a loss of responsiveness to the signals transduced by regulatory proteins; for example, if CRP has an inappropriately high affinity for a gene promoter, it will continue to stimulate transcription potentially long after the cell has satisfied its energy needs. Conversely, if a protein has low affinity for a DNA site, it will move on and spend more time elsewhere on the chromosome. Therefore, all gene promoters are fine-tuned by natural selection to have a useful balance between protein binding and dissociation (except perhaps in intracellular symbionts, which can lose gene regulatory functions because they live in static environments).

Protein affinity for a DNA site is usually expressed as a dissociation constant, Kd (the lower case “d” should be a subscript capital “D”). A “dissociation” constant is used because it is technically difficult to measure how quickly a protein binds to DNA, whereas it is much more straightforward to measure how long it takes for a protein to let go after it has bound to a target site. Bandshift analysis (which we use in the lab) is the most popular technique for calculating Kd. The Kd value is expressed as the molar concentration of protein needed to bind, and thus shift, half of the radioactive “bait” DNA in a binding reaction. For example, 1 nM of CRP can shift half of the DNA when the mglBAC promoter is used as bait because this promoter contains a high-affinity CRP site. Therefore, for the mglBAC promoter, CRP's Kd = 1 nM. On the other hand, it takes 100 nM of CRP to shift bait DNA that contains a less favourable binding site.

In a bandshift assay, a purified protein (in my case CRP) is mixed with a low concentration of bait DNA containing a target site to which the protein is expected to bind, in addition to a very high concentration of "bulk" DNA for which the protein is expected to have very low affinity. Under these reaction conditions, the protein will spend much of its time bumping into DNA sites with which it can’t form lasting associations. Even though the protein will rarely bump into a bait DNA molecule, if it has a high affinity for a site in the bait DNA, it will hold on and not let go. If the bait DNA contains a low affinity site, more protein molecules are needed in the reaction so that as soon as a protein molecule lets go of the binding site, another protein molecule is likely to quickly bump into that same site and hold on, albeit for a short time.

I recently tested the binding affinity of both E. coli CRP and H. influenzae CRP for a perfect CRP binding site called “ICAP”. Both proteins demonstrated an identical affinity for the site. Because this was an unexpected result, further thinking revealed that the reaction conditions I used were not suited to detect super-high affinity. I realized that to detect super-high affinity, I would need to decrease the amount of bait DNA in my reactions. At the concentration of bait DNA used, protein with only a high affinity (as opposed to a super-high affinity) for the site could also readily shift half of the bait DNA. In other words, there was so much bait DNA in the reaction that I had to load more protein than should be necessary to shift even half of the DNA. This concentration of bait DNA has been convenient for testing low to high affinity DNA sites, but is not sensitive to super-high affinity. Thus, I will need to redo the experiments with vanishingly small (pM) amounts of protein and bait DNA.


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