Looking forward…

I was really happy with the protein I was able to synthesize, the experiments I was able to conduct and the results I received! By analyzing these results, I have been able to come up with new ideas for future experiments we can conduct by incorporating different UAAs into GFP. Continuing with the 3FY work done this summer, I would like to incorporate other fluortyrosines including di- and tri- fluorotyrosines. I would conduct similar titration experiments to see if the pKa shifts through the addition of fluorines. Finally, I will conduct similar fluorescence spectral analysis on the ortho-nitrobenzyl tyrosine GFP as well.

[Read more…]

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.

[Read more…]

Glaser-Hay Conjugation Results

After 7 weeks of research I had finally reached a point where I would be able to conduct experiments on the green fluorescent protein I had synthesized and collect results! I began working with the p-propargyloxyphenylalanine (pPrF) GFP as I had synthesized that successfully first. The unnatural amino acid pPrf is synthesized using the starting material tyrosine and propargyl bromide. Once the two are reacted, the hydrogen on the tyrosine side group oxygen is pushed off and the oxygen is now bonded to a propargyl group. The propargyl group, which contains a C-C triple bond, introduces a functionality which is not naturally present in amino acids and proteins. This allows us to conduct alkyne reactions with specificity. In order to understand the properties of the fluorophore, I set up an experiment where the UAA would be reacted with alkyne molecules. This unique reaction is known as the Glaser-Hay Reaction and allows us to create artificial polyynes. After the Glaser-Hay reactions were conducted with various alkyne molecules, emission spectra were collected for each of the reactions. We were able to see significant shifts in fluorescence peaks with the various molecules which meant that the weeks of work had paid off with some very interesting results!

[Read more…]

Goals with GFP with Unnatural Amino Acids

In my last post I discussed how my primary goal for the summer was to synthesize different types of GFP, all with some unnatural amino acid (UAA) inserted into the protein sequence. The UAAs that I inserted into GFP were 3-fluorotyrosine (3FY), ortho-nitrobenzyl tyrosine (ONBY), and p-propargyloxyphenylalanine (pPrF). The goal of inserting these UAAs into the protein is to observe how they modify the chemical properties of the GFP fluorophore. When analyzed with a spectrophotometer, wild-type GFP has a characteristic spectrum. Because the UAAs are inserted in the fluorophore region of the peptide, a shift in the fluorescent spectrum is apparent in GFP with UAAs incorporated. The next step in this project was analyze the protein itself using spectroscopy and conducting experiments on the protein to see if there is a shift in the fluorescence spectra.

[Read more…]

Synthesizing Green Fluorescent Protein with Unnatural Amino Acids

The first task of the summer was synthesizing and purifying green fluorescent protein (GFP) with various unnatural amino acids. The unnatural amino acids (UAAs) that were of interest were 3-fluorotyrosine (3FY), ortho-nitrobenzyl tyrosine (ONBY), and p-propargyloxyphenylalanine (pPrF). The first step in the synthesis of GFP with the UAAs is inserting the genes that code for GFP (either TAG-66 or Waldo) and the genes for the enzyme that inserts the UAA into the protein (a synthetase) into E. coli by electroporation. The transformation of the bacteria with the plasmids was verified by plating the cells onto agar containing the antibiotics ampicillin and chloramphenicol. Since the plasmids contain antibiotic resistance genes, only the cells that have been successfully transformed will grow on the plates. After this, colonies are transferred from the plates into culture vials and induced with IPTG/arabinose and the desired UAA. Once the cells have grown and expressed the protein overnight, the protein is extracted from the cells and purified. To verify that the cells have expressed the protein of interest and that it has been successfully purified, the protein sample is run on a gel by gel electrophoresis.This process has led to some unexpected challenges as often times you are unable to visualize the protein on the gel.  Some issues faced throughout this process include unsuccessful transformation of the bacteria, phage infections in some of the culture media and plates, insufficient expression of the protein, and failure to synthesize the UAA. After 5 weeks of trial and error, I was finally able to obtain GFP with the UAAs of interest.