BBa_K2689000

pHluorin2 is a ratiometric, pH-dependent GFP. Its 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.

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.

Absorption maximum 1: 395nm

Absorption maximum 2: 475nm

Emission maximum: 509nm

You can find the original paper by Matthew Mahon here.

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/417nm and plotted it on the following graph.

While the relationship does not seem to be linear at least in our chosen pH range a pretty good estimate of the pH can be calculated using a linear approximation.

BBa_K2689001

Syntaxins are nervous-specific integral membrane proteins involved in membrane fusion. They possess a C-terminal transmembrane domain, a SNARE domain and an N-terminal regulatory domain. Syntaxin-1A is involved in ion channel regulation and synaptic exocytosis. It interacts with SNAP25 and Synaptobrevin-2 to form the SNARE complex. This complex formation brings vesicle membrane and cell membrane into close proximity, which leads to merging of both membranes. In neuronal cells, synaptic vesicles store neurotransmitters that are released upon stimulation of the cell. This exocytosis process is, among other things, is regulated by Syntaxin1a. Syntaxin-1A is a known substrate of botulinum neurotoxin C, a protein with metalloprotease activity which prevents the exocytosis of neurotransmitters into the neuromuscular junction. Consequently, this leads to flaccid paralysis symptoms of the illness botulism.

BBa_K2689002

Synaptosomal-associated Protein 25 (SNAP-25) is a t-SNARE protein involved in the regulation of membrane and vesical fusion. It is integrated into the membrane via palmityl side chains and faces the cytosolic side. Together with Syntaxin-1A and Synaptobrevin-2, it forms the SNARE complex, in which it contributes 2 a-helices. This complex brings together vesicle membrane and cell membrane, which leads to the fusion of both. In neuronal cells, this leads to the exocytosis of neurotransmitters into the interneuronal junction. SNAP-25 is a known substrate of botulinum neurotoxin A, C and E, a protein with metalloprotease activity which prevents the exocytosis of neurotransmitters into the neuromuscular junction. Consequently, this leads to flaccid paralysis symptoms of the illness botulism.

BBa_K2689003

Omomyc is a dominant-negative mutant of Myc, a family of proto-oncogenes encoding for transcription factors which regulate growth, proliferation, tumorgenesis, and apoptosis. Myc dimerizes with Max over its basic helix-loop-helix zipper domain. This heterodimer then binds to the DNA enhancer box (E-Box) sequence and thus regulates the expression of corresponding genes. In cancer Myc often is constitutively expressed, leading to increased proliferation signals and thus to excessive tumor growth. Omomyc possesses a mutated basic helix-loop-helix zipper domain, in which 4 amino acids were exchanged: S57T, E64I, R70Q and R71N. These mutations lead to an altered dimerization behavior of the protein. Omomyc efficiently forms homodimers and furthermore also forms heterodimers with wild-type Myc, leading to a decreased formation of Myc/Max dimers. Accordingly, Omomyc works as a suppressor of Myc/Max binding to E-box elements and leads to a suppressed activation of artificial E-box promoter elements. In the past it was shown that Omomyc can selectively trigger apoptosis in cells overexpressing Myc, however the exact mechanism remains unclear. Researchers proposed that this happens through the transcriptional repression of specific genes. Through its ability to induce apoptosis in cells overexpressing Myc, as various cancers do, Omomyc may be subject to new therapeutic opportunities in the future.