Detection of Internal Cellular pH using Enhanced Yellow Fluorescent Protein (EYFP)

Fluorimeter
Nanoscience Chemistry
Advanced Inorganic Lab
Bacterial Fluorescent Protein

Fluorescence Microscopy. E.coli strain W3110
containing a TetR-EYFP plasmid visualized under a confocal microscope at Ohio State University.

The ability to use the green fluorescent protein (GFP) as a molecular probe and biosensor initiated a new period in the study of cell biology. Before the advent of GFP fusions, small fluorescent molecules were used to label live cells in order to monitor cellular processes. However, these molecules tended to be highly phototoxic and had to be “linked” to the cell through various procedures. Unlike these fluorescent molecules, the autofluorescent GFP, which does not rely on molecular cofactors to induce fluorescence, is harmless to the cell. Because the fluorescent protein is fused to a protein already present within the cell, the extra “linking” procedures necessary for small fluorescent molecules do not have to be carried out. This allows the researcher to monitor cellular processes in the cells without harming and disrupting the cells of interest.

Various fluorescent proteins developed by Roger Tsien (from Tsien lab)

The recent creation of color-shifted genetic derivatives of GFP, such as blue (BFP), cyan (CFP) and yellow (YFP), has enabled a more thorough study of both eukaryotic and prokaryotic cellular processes. These proteins are most commonly used in cellular imaging experiments using confocal or widefield microscopy. When coupled with microscopy, the variety of fluorescent proteins also allows for the use of fluorescence resonance energy transfer (FRET), a technique that allows for the detection of the precise location and nature of the interactions between specific molecular species in living cells.

These various derivatives differ not only in the color of their emitted light, but also in their photochemistry. The fluorescence signal of these proteins can be affected by chloride or calcium ion concentration as well as pH. For example, the fluorescence signal of CFP is not pH dependent, meaning you will get a similar signal when the protein is both protonated and deprotonated. However, YFP is highly pH dependent, thus when the protein is deprotonated the fluorescence signal is much higher than when the protein is protonated. The difference in photochemical properties among the fluorescent proteins allows for the study of ion concentration and localization with in the cell as well as internal cellular pH.

In Dr. Slonczewski’s lab, we transformed E.coli strain W3110, a strain continually selected for acid resistance, with a plasmid containing TetR-EYFP (enhanced yellow fluorescent protein). The pH dependence of EYFP and the plasmid’s location within the cytoplasm of the cell allows us to detect changes in the cell’s internal pH using the Fluormax-3.

In order to affirm the pH dependence of EYFP in live W3110 cell culture, these cells were resuspended in media that was pH adjusted to 5.5, 6.5, 7.0 and 8.0. This media contained 10mM benzoate, a proton uncoupler that allows protons to pass through the membrane by equalizing internal and external pH. Both excitation and emission spectra were taken using the following parameters:
Excitation: excitation ? 450-520 peak at 514 (slit width of 2)
emission ? 550 (slit width 20)
Emission: emission ? 500-700 peak at 527 (slit width 4.0)
excitation ? 480 (slit width 4.0)
The pH dependence of the yellow fluorescent protein in vivo is evident when stacking the individual spectra. Both the emission and excitation spectra exhibit a clear gradation between pH 5.5, 6.5, 7.0, and 8.0 (figure 1, figure 2).

For future research using the Fluromax-3, we are working on developing a method to test the recovery of the cell’s internal pH by using a fluorescent time course. Continuous spectra will be taken of cells suspended in pH 7.5 buffered media, the pH of
the media will then be shifted immediately to pH 5.5 using HCl and the Fluromax-3 injection port. During this time the Fluromax-3 will continue to read spectra over a 10 min time period, allowing us to view the recovery of the cell’s internal pH over time. Thus, this method would allow for the visualization of pH homeostasis within the cell.
Published by undergraduate: Wilks et al, 2007.

Figure 1. Excitation spectra of EYFP. E.coli strain W3110 containing a TetR-EYFP plasmid was grown for 14 hours overnight in Luria-Bertani medium containing 100 mM KCl (LBK). The culture was spun down and resuspended to OD600 of 0.6 in pH adjusted minimal media containing 10mM benzoate

Figure 2. Emission spectra of EYFP. E.coli strain W3110 containing a TetR-EYFP plasmid was grown for 14 hours overnight in Luria-Bertani medium containing 100 mM KCl (LBK). The culture was spun down and resuspended to OD600 of 0.6 in pH adjusted minimal media containing 10mM benzoate.