Friday, November 30, 2007

Success with equations

I have solved the issue of excess versus limiting DNA in bandshift reactions. It turns out that my confusion arose because of the two distinctly different ways that the equation for deriving equilibrium binding constant can be derived: the common (overly simplified) equation for calculating Kd (the dissociation constant, which reflects a protein’s affinity for a DNA site) ignores the important qualifiers of the protein and DNA concentrations in the reaction. Unfortunately, the Kd equation cannot be easily illustrated in this blogger post due to my inability to write equations in a blog, so I will add the equation (and its derivatives) tomorrow when I have time to draw them out and edit this post.

The bottom line is that all of the bandshift experiments I have conducted have been informative, and now I can confidently proceed to the next step of measuring accurate Kd values for CRP binding to different CRP sites. Also, tomorrow we will test a second prep of His-tagged Sxy to see if once again CRP has been co-purified along with Sxy. If positive (ie. CRP was pulled down with His-Sxy), we can easily test how much salt needs to be added to His-Sxy to prevent the co-purification of CRP; the amount of salt needed to block the interaction will give us a sense of the strength of affinity between the two proteins.

Thursday, November 22, 2007

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.

Sunday, November 11, 2007

More results from Sunita and Andrew

Our results that suggest Sxy binds to DNA are exciting but suspicious; all Sxy-DNA binding data can be explained by the presence of contaminating CRP in the Sxy protein preps. This is because the Sxy-DNA binding data is identical to CRP-DNA data. First, EcSxy binds DNA but HiSxy does not. Second, EcSxy greatly prefers the pilA-N (CRP-N mutant) site over the wildtype pilA CRP-S site. Third, when EcSxy and EcCRP are mixed together, only one protein binds to a DNA molecule, suggesting that both proteins target the same site (this is consistent with the pilA-N data).

The next set of experiments is clear: 1) Test whether EcSxy can bind DNA in the absence of cAMP (EcCRP cannot), 2) Test whether EcSxy binds to a pilA promoter that lacks its CRP site, 3) Use western blots to probe for EcCRP in the Sxy preps, and 4) Test DNA binding by EcSxy that has been isolated from a crp- expression strain.

However, several arguments can still be made that EcSxy does in fact bind DNA. First, EcSxy and HiSxy were isolated form the same E. coli strain using the same procedure, thus we would expect EcCRP to contaminate the HiSxy preps as well (which clearly has not happened because HiSxy preps don’t bind DNA). Second, far-western analysis has not detected EcCRP in the Sxy preps.

If tomorrow’s experiments show that EcSxy binds DNA in the absence of cAMP, two new hypotheses need to be addressed: 1) Does EcSxy prefer the pilA-N promoter not because it binds the CRP-N site, but because the CRP-N site makes DNA more bendable than the wildtype CRP-S promoter? 2) Does HiSxy fail to bind DNA because bandshift reaction conditions are not favourable for H. influenzae proteins?

Thursday, November 08, 2007

Hot stuff (aka. sticky Sxy)

After a grueling few days of bandshift experiments to study CRP and Sxy binding to the pilA promoter, we have made the excellent discovery that Sxy binds to DNA. Surprisingly, it appears that Sxy may bind to the CRP site in this promoter. We have several promoter constructs that lack various parts of the wildtype promoter - we can use these to directly test whether CRP and Sxy bind to the same site. We are taking a break from bandshifts this afternoon to analyze the data and plan our next set of experiments. We will begin to frame these results as a figure for our DNA-binding manuscript.