Team:METU Turkey/Results Discussion/ITF


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Our protein, that is CooA transcription factor, has two tryptophan residues in its structure. DNA binding to protein can cause conformational change in the protein structure so tryptophan orientation changes in the protein. By carrying out intrinsic tryptophan fluorescence, we expected to observe fluorescence change in tryptophan residues with binding of CooA to pCooF. We used CooA in three different forms that are inactive or oxidized, reduced by DTT and reduced –CO bounded. On the other hand, we used only one DNA template that is pCooF. We carried out the experiment two different buffers. We prepared for oxidized protein and this buffer contained every component in reaction buffer except reducing agents such as DTT,sodium dithionite and also bovine serum albumin. Unlike oxidized protein, reduced CooA is needed reducing agents but because of giving fluorescence of tryptophan, BSA was not added to solution.

After prepared the buffers, we started to measure our sample via titrating CooAs with the pCooF. Measurements are taken in this ratio of protein: DNA that are 1:1; 1:2; 1:3; 1:4. On the other hand, we made negative control to ensure intensity decrease resulting from dilution or time effects. We excited CooA at 280 nm and we took emission scan at 300-400 nm. Our slit number was 4 nm that is 2 turn. Each reaction happened at room temperature for 20 minutes. We took 20 emission scans. We observed decrease in the intensity of the fluorescence; however when looked at negative control measurements, we saw the decrease in intensity because of the dilution and time effects. So we cannot say that that reduction is caused from pCooF – CooA interaction. Moreover, we suspect that heme group in the CooA quench fluorescence of the tryptophan and also heme group gives absorbance at 280- 400 nm so our protein might give the fluorescence at that range itself.
yellow : red/Co 28 nM pcoof 20 .min

pink:red/ CO + buffer

blue: red/ CO 56 nM pcoof 40. min

green: NC red/ co 10 ul elution buffer 20.min

purple: NC red/ CO 20 ul elution 40. min
white :oxizied.+ 56 nM pCoof + 40. min

blue : red / CO + 56 nM pCooF + 40 min