3FY Titration Results

Excited by the successful results of the p-propargyloxyphenylalanine GFP Glaser-Hay fluorescence results, I moved on to studying the 3-fluorotyrosine (3FY) GFP I had synthesized earlier. This was a project that had be started earlier by a student who had graduated this past semester so I was thrilled to take it on. 3-fluorotyrosine is another amino acid that excites me as the fluorine groups can significantly modify the chemical properties of the protein. In particular, I decided to look at the effects of 3FY on the pKa of the protein. This was done by conducting titration experiments on the 3FY-GFP and wild-type (WT) GFP.  Titration experiments involve adding an acid or a base to protonate or deprotonate a compound. Emission spectra can tell us about the amount of protein that is protonated or deprotonated as the two forms emit different wavelengths of light. The protonated peak for 3FY-GFP occurs around 450 nm while the protonated peak is at around 525 nm. By calculating the ratio of the protonated peak to the deprotonated peak, we can determine what percentage of the protein is protonated and deprotonated. A baseline spectra was acquired for both 3FY-GFP and wild-type GFP. Subsequently, acid or base was added to the sample in small intervals followed by another emission reading. This was repeated several times to achieve a full titration curve. As acid is added to the sample the ratio of the protonated to deprotonated peaks went up while the reverse was true for base additions. We observed significant changes in the pKa of the 3FY as compared to WT-GP when the titration curves were plotted.

Exciting times, huh? Thanks for reading!

Comments

  1. relevymyers says:

    does this imply that under UV light 3FY-GFP emits a different fluoresce color than WT-GFP. If so that would be really cool because you could fuse each one to a different protein in the same cell and look at each protein separately under a different wavelength in the same cell