Part:BBa_K2689000
pHluorin2
pHluorin2 is a ratiometric, pH-dependent GFP. It's excitation spectrum varies as the pH increases/decreases. This allows pHluorin2 to be used as an accurate biosensor. A special use case is the tracking of proteins that move between different cell compartments and encounter varying pH environments. Compared to pHluorin, pHluorin2 additionally shows higher fluorescence levels. It was developed by Matthew Mahon.
Usage and Biology
Biosensors are great tools for biologic research. GFP has been extensively used in the past for studying protein dynamics in the cell. For those purposes it is usually fused to another protein which subsequently can be tracked via fluorescence microscopy.
While GFP itself shows a relatively stable fluorescence behavior across a wide pH range (at least too stable to be quantified easily), a look at the chromophore suggests that quite a lot of acid-base chemistry is influencing the fluorescence. Because those reactions are confined to the inner core of GFPs beta barrel structure, the solvent pH does not affect the chromophore in a easily quantifiable way. The idea behind pHluorins is to exploit these characteristics and change the structure of GFP in a way that it interfaces the internal reactions to the solvent pH so that it has a direct influence on their fluorescence behavior. For this purpose Miesenböck, et al. performed a directed mutagenesis approach in which they developed two forms of pH-dependent GFPs: Ecliptic and ratiometric pHluorin.
These pHluorins show a marked difference in their absorption spectra when encountering different ambient pH. The wild type form of GFP has a bimodal excitation spectrum with two peaks at about 395nm and 475nm. While the ecliptic variants of pHluorin show a decreased fluorescence signal with lower pH, the ratiometric pHluorins show a more complex pattern: With lower pH, the absorption at 395nm decreases while the absorption at 475nm increases. This is a key attribute for using it as a fluorescent pH probe.
When carrying out protein tracking experiments with pHluorin one has no ability to differentiate the cause for a decreasing fluorescence signal. While a possible reason can be a decrease pH, several other effects influence the signal strength. Those can be changing protein concentrations (e.g. caused by degradation), or fluorescence quenching due to long exposure times. To actually identify a decrease in pH, a single signal value is not enough. This is where ratiometric proteins come into play. Rather than using just one signal measurement to estimate the ambient pH, the observer performs two measurements at a different wavelength. Because the preferred excitation wavelength of pHluorin changes with pH, the ratio of the signals is an indicator of pH unaffected by the confounding effects described above.
Sometimes pH quantification using pHluorin can still be difficult when concentration of it is too small to produce reliable measurements. This is the case, if pHluorin is coupled to another protein with low expression count, e.g. parathyroid hormone 1 receptor (PTH1R).
Luckily there are other improvements to GFP apart from making it a pH biosensor. EGFP is a commonly used variant of GFP which contains two mutations that improve its fluorescence characteristics. One of those is the F64L mutation that improves correct protein folding and therefore leads to a higher signal. The other is S65T (a direct modification to the chromophore) that quenches the 395nm absorption in favor of the 475nm absorption. As only the first mutation carries a desired effect it is the only one that is included in pHluorin2.
In summary pHluorin2 is a GFP with the pH-dependent fluorescence characteristic of pHluorin and the superior efficiency of EGFP making it an ideal tool for studying endocytotic pathways in cells.
Important Data:
Absorption maximum 1: 395nm
Absorption maximum 2: 475nm
Emission maximum: 509nm
You can find the original paper by Matthew Mahon in the references.
Characterization of BBa_K2689000
For measuring pHluorin2 we cloned our sequence in a pET28a vector and transformed it into electrocompetent Lemo21 E. coli cells. After inducing a 1.8L culture with IPTG and incubating at 20°C for 18 hours, cells were harvested and lysed by sonification. Following centrifugation, the lysate was purified using a GE Healthcare HisTrap FF column.
For the fluorescence measurement, we had the purified protein in PBS with a buffer capacity of 20uM ready. We analogously prepared a PBS buffer with a buffer capacity of 70uM and titrated it to pH values from 5.3 to 7.5. 80ul aliquots of the second buffer were given into a White Opaque PerkinElmer CulturPlate-96. For a given pH three samples were loaded into three wells. After that, we added 20ul of our protein solution (with a concentration of about 0.5mg/ml). We measured fluorescence using a Tecan M200 Infinite Pro plate reader with the following settings: Excitation Scan Excitation Wavelength Start 340nm Excitation Wavelength End 520nm Excitation Wavelength Step Size 3nm Emission Wavelength 550nm (Note: This is not the absorption maximum. However, fluorescence can readily be measured at 550nm even though with a lower signal strength. We did that to avoid measuring the signal emitted by the plate reader). Integration Time 20us Number of flashes 25 Gain 50
You can find the spectra (averaged over each three wells) at different pH values below
You can see the effect on the two absorption maxima even better when plotted for each pH:
From our measurements, we calculated the ratio of 415nm/478nm and plotted it on the following graph.
References
Matthew J. Mahon (2011) pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein - https://dx.doi.org/10.4236%2Fabb.2011.23021
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 747
- 1000COMPATIBLE WITH RFC[1000]
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