ABB  Vol.2 No.3 , June 2011
pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein
ABSTRACT
Green florescent protein (GFP) variants that are sen-sitive to changes in pH are invaluable reagents for the analysis of protein dynamics associated with both endo- and exocytotic vesicular trafficking. Ratiomet-ric pHluorin is a GFP variant that displays a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidi-fication, pHluorin excitation at 395 nm decreases with a corresponding increase in the excitation at 475 nm. GFP2, a GFP variant that contains mammalian-ized codons and the folding enhancing mutation F64L, displays ~8-fold higher florescence compared to pHluorin upon excitation at 395 nm. Using GFP2 as a template, an enhanced ratiometric pHluorin (pHluorin2) construct was developed to contain fully mammalianized codons, the F64L mutation and ten of the thirteen pHluorin-specific mutations. As a result, pHluorin2 displays markedly higher flores-cence when compared to pHluorin while maintaining the ratiometric pH-sensitivity. Unlike native pHluorin, pHluorin2 expressed in the ligand-binding domain of the parathyroid hormone 1 receptor is readily detectable by confocal microscopy and dis-plays a marked increase in florescence upon ligand-induced endocytosis to intracellular vesicles. Thus, pHluorin2 displays enhanced florescence while sustaining ratiometric pH-sensitivity, representing a significant improvement for this methodological ap-proach.

Cite this paper
nullMahon, M. (2011) pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein. Advances in Bioscience and Biotechnology, 2, 132-137. doi: 10.4236/abb.2011.23021.
References
[1]   Shaner, N.C., Patterson, G.H. and Davidson, M.W. (2007) Advances in fluorescent protein technology. Journal of Cell Science, 120, 4247-4260. doi:10.1242/jcs.005801

[2]   Ciruela, F. (2008) Fluorescence-based methods in the study of protein-protein interactions in living cells. Cur- rent Opinion in Biotechnology, 19, 338-343. doi:10.1016/j.copbio.2008.06.003

[3]   Miesenbock, G., De Angelis, D.A. and Rothman, J.E. (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature, 394, 192-195. doi:10.1038/28190

[4]   Llopis, J., McCaffery, J.M., Miyawaki, A., et al. (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Pro- ceedings of the National Academy of Sciences USA, 95, 6803-6808. doi:10.1073/pnas.95.12.6803

[5]   Awaji, T., Hirasawa, A., Shirakawa, H., et al. (2001) Novel green fluorescent protein-based ratiometric indi- cators for monitoring pH in defined intracellular micro-domains. Biochemical Biophysical Research Communi- cations, 289, 457-462. doi:10.1006/bbrc.2001.6004

[6]   Bizzarri, R., Arcangeli, C., Arosio, D., et al. (2006) De- velopment of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies. Bio- physics Journal, 90, 3300-3314. doi:10.1529/biophysj.105.074708

[7]   Heim, R., Cubitt, A.B. and Tsien, R.Y. (1995) Improved green fluorescence. Nature, 373, 663-664. doi:10.1038/373663b0

[8]   Sankaranarayanan, S., De Angelis, D., Rothman, J. E., et al. (2000) The use of pHluorins for optical measurements of presynaptic activity. Biophys Journal, 79, 2199-2208. doi:10.1016/S0006-3495(00)76468-X

[9]   Tawfeek, H.A., Qian, F. and Abou-Samra, A.B. (2002) Phosphorylation of the receptor for PTH and PTHrP is required for internalization and regulates receptor signa- ling. Molecular Endocrinology, 16, 1-13. doi:10.1210/me.16.1.1

[10]   Thastrup O.T.S., et al. (1995) Fluorescent Proteins. US patent number: 7314915.

[11]   Ferrandon, S., Feinstein, T.N., Castro, M., et al. (2009) Sustained cyclic AMP production by parathyroid hor- mone receptor endocytosis. Nature Chemical Biology, 5, 734-742. doi:10.1038/nchembio.206

 
 
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