I am running fluorescence spectroscopy on a protein with two endogenous tryptophan residues and comparing the fluorescence with that of a site-directed mutant of the same protein, in which both tryptophan residues have been substituted for other aromatic residues (1 Phe and 1 Tyr). However, the results show that even with the buffer subtracted and the fluorescence signal normalized (ie. corrected for differences in the concentration of the samples scanned) the wild type protein and the mutant with no Trp residues give the same same fluorescence signal when excited at 295nm. This seems counter intuitive, as I expected that the mutant with no tryptophan residues would have little to no fluorescence signal at that wavelength. The samples of the protein were purified by IMAC and desalting and sequencing has previously confirmed the presence of the desired mutations in the site-directed mutant. That being said, the likelihood that a contaminant is responsible for both signals seems low, as for them to be essentially exactly the same they would have to make up the majority of the protein in the sample. However, western blot and SDS-PAGE have already confirmed that this is not the case. Has anyone encountered a similar problem before or does anyone know of any possible cause for absorption at 295nm in a protein sample with no tryptophans?
The tyrosine can also emit in this range, just at a slightly lower intensity. However buffer conditions may enhance this signal
Let me know the peak positions of both Wild type and mutant fluorescence spectra. Then I would be able to explain the details cause of this problem.
I do not know this protein but in the wild type, the contribution of trp to fluorescence could be low if trp are located on (or near) the protein surface (e.g. how many other tyrosine are in the protein) .
Maybe you can estimate the accessibility of your trp with a fluorescence quencher such as iodide or acrylamide and the Lehrer and the Stern-Volmer plots. This is, I think, easier to do than the enzymatic digestion of the protein.
Do you clearly see a fluorescence peak for trp (e.g. did you made the whole spectra or a single measurement after excitation at 295 nm) because if you have a problem with the bandwidth of you fluorimeter, it can be only due to autofluorescence.
Thanks for the suggestions everyone. In regards to your question Basir, the peak in the emission spectrum when excited at 295nm is at 340nm for both the wt and Trp knockout version of the protein and the intensity of emission is equivalent (both ~14A.U).
Might consider the formation of tyrosinate, which has the max. absorbance at 295 nm and emission at 345 nm. The ionic form of tyrosine in proteins is typically observed in alkaline solution, but has been reported in neutral condition.
Does the change in molar absorptivity of the protein dissolved in spectroscopic grade GuHCl between the wt and mut accurately reflect the mutations you expect? You can make this measurement with a good UV/VIS instrument and quality quartz cuvette. The difference should be dramatic, so this is a quick and inexpensive way to confirm you have what you believe you have. The second possibility is a background fluorescence issue, which are often difficult to control for if you are working with very dilute protein solutions and have fluorescent contaminants or even a non-fluorescent, but UV absorbing background. If you have, for instance, some other 295 nm UV-absorbing species (many have broad UV absorption spectra), this will block the fluorescence excitation light and attenuate the emission fluorescence signal from your protein...known as the "inner filter effect." Other comments re: weakly fluorescent residues are also relevant. Amino acid analysis can be used to confirm the relative residue content--always good to double check to make sure you have what you believe you have.
Have you mutated both the tryptophans (double mutant), because at 295 nm excitation the probability of Y and F fluorescence (quantum yield) is very less compared to W. If one of the 2 W is not mutated there might be a probability of emission at 340 nm. Just cross check with your sequence once again.
The intensity, quantum yield, and wavelength of maximum fluorescence emission of tryptophane is very solvent dependent. The fluorescence spectrum shifts to shorter wavelength and the intensity of the fluorescence increases as the polarity of the solvent surrounding the tryptophane residue decreases. Tryptophan residues which are buried in the hydrophobic core of proteins can have spectra which are shifted by 10 to 20 nm compared to tryptophans on the surface of the protein. Tryptophan fluorescence can be quenched by neighbouring protonated acidic groups such as Asp or Glu.
Dear Tyler Auld
As you know intrinsic fluorescence of protein is mainly due to Trp and Tyr. When we excite at 280 nm e protein containing only Tyr gives emission around 305-310 nm but never at 340 nm. On the other hand protein containing Trp can emit anywhere between 320-360 nm depending on exposure of the Trp to the solvent (polar). Moreover, tyr hardly excites at 295 nm. When excitation of 295 nm is used the fluorescence is mainly due to Trp. Therefore, It seems that your protein is either contaminated with some other protein/peptide containing Trp. This is not the case because you have already checked the purity. This means your protein surly contains Trp (s). If you are confident with your DNA sequencing confirm it by Mass spectroscopy or go for protein sequencing.
Thanks
I agree with Basir. It looks that there are Tep in your sequence or contaminating the sample. Both the wt and the mutant proteins cannot give the same spectra. How big is your protein, can you do mass spec and confirm?.
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