The Effect of pH on Green Fluorescent Protein: a Brief Review

Tessa N. Campbell and Francis Y.M. Choy

Center for Environmental Health, Department of Biology University of Victoria Victoria, British Columbia, Canada. Address correspondence to: Tessa N. Campbell               Department of Biology, University of Victoria, Box 3020 STN CSC Victoria, British Columbia V8W 3N5 Canada Tel.: (250) 721-7138 Fax: (250) 477-0465 e-mail: tessac@uvic.ca   Key words: GFP, pH, fluorescence

Green fluorescence demonstrating successful expression of the lysosomal hydrolase glucocerebrosidase (EC 3.2.1.45) fused to EGFP in Clontech's pEGFP-N1 vector in two HeLa cells 48 hours after transfection (source: T.N. Campbell, unpublished results).

Abstract

Introduction

Effect of pH on GFP

Uses of GFP to study processes in different pH environments

GFP as a pH indicator

Conclusions

Aknowledgements

References
 
 

Green fluorescent protein (GFP) is rapidly becoming one of the most frequently employed molecular reporters. Its use in monitoring gene expression and protein localization has been well documented. Different mutational approaches have created numerous GFP variants with optimized expression, differing spectra, and differing pH sensitivity. This last characteristic, though still poorly understood mechanistically, has attracted an increasing amount of interest. To date, GFP variants have been developed with pKas ranging from 4.8 (T203I) to 8.0 (YFP-H148G/T203Y). The objective of this review is to outline both the effect of pH on GFP fluorescence and the uses of GFP to study processes in different pH environments.
 

It is becoming increasingly difficult to peruse a biological, microbiological, or biochemical journal and avoid encountering the phrase "green fluorescent protein". The existence of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria was reported decades ago (Shimomura et al. 1962, Morise et al. 1974). However, the cloning (Prasher et al. 1992) and heterologous expression of its cDNA (Chalfie et al. 1994) soon ignited an explosion of applications for GFP. Such applications include monitoring of gene expression (Li et al. 1999, Takeuchi et al. 1999, Wheeler et al. 2000), protein localization (Wang and Hazelrigg 1994, Kaether and Gerdes 1995, Lim et al. 1995, Harada et al. 2000), host-pathogen interactions (Dhandayuthapani et al. 1995, Valdivia et al. 1996), cellular dynamics (Rizzuto et al. 1995, Gerdes and Kaether 1996, Fricker et al. 1999), protein purification (Cha et al. 1999, Dabrowski et al. 1999), Ca²⁺ concentration (Miyawaki et al. 1997, Romoser et al. 1997, Allen et al. 1999), and pH levels (Kneen et al. 1998, Llopis et al. 1998, Miesenböck et al. 1998, Robey et al. 1998).

Encoded wild-type GFP is 238 amino acids and approximately 27 kD (Prasher et al. 1992). It absorbs maximally at ~393 nm with a minor peak at 473 nm, and emits green light at 509 nm (Ward et al. 1980). Different mutational approaches have optimized expression by altering the promoter, codon usage, or ribosome binding; by eliminating splicing; or by enhancing folding. GFP variants with differing spectra have also been created, permitting multicolor microscopic visualization (Rizzuto et al. 1996, Palm and Wlodawer 1999). GFP fluorescence is due to the presence of a chromophore intrinsic to the primary structure, thus requiring no additional cofactors. The chromophore is a p-hydroxybenzylideneimidazolinone formed from Ser⁶⁵-Tyr⁶⁶-Gly⁶⁷. Fluorescence is acquired through the creation of the imidazolinone by nucleophilic attack of the amino group of Gly67 on the carbonyl group of Ser65, followed by dehydration, and then by oxidation of the hydroxybenzyl side chain of Tyr66 by atmospheric oxygen. The beta-can crystal structure of wild-type GFP consists of an 11-stranded beta-barrel with an alpha-helix running up the axis of the cylinder. The chromophore is attached to the alpha-helix and buried in the center of the cylinder (Ormö et al. 1996, Tsien 1998). The in vitro spectral properties of GFP are influenced by temperature, ionic strength, protein concentration, and pH (Ward et al. 1982). Recently, interest in the pH sensitivity of GFP has led to the successful introduction of GFP as a noninvasive pH indicator. This article briefly outlines the effect of pH on GFP fluorescence and the uses of GFP to study processes in different pH environments.

The fluorescence of wild-type GFP (wtGFP) is stable from pH 6-10, but decreases at pH<6 and increases from pH 10-12 (Ward 1981, Patterson et al. 1997). A number of studies have demonstrated that different GFP variants display greater pH sensitivity (Kneen et al. 1998, Haupts et al. 1998, Elsliger et al. 1999). In denatured wtGFP, the chromophore has pH-dependent spectral characteristics due to the ionization of the Tyr⁶⁶ phenolic group (Ward et al. 1982). The phenolate form of the chromophore absorbs maximally at 448 nm, compared with that of 384 nm for the uncharged phenol. The pKa for this transition is 8.1 (Ward et al. 1980). Based on spectroscopic examination of the excited state dynamics of GFP, Chattoraj et al. (1996) proposed that wtGFP exists in one of two ground states, A and B, which differ in the protonation state of the chromophore. An excited-state proton transfer reaction rapidly converts state A to an intermediate state I, which is then slowly converted to state B. The existence of two ground state conformations, each with distinct spectral characteristics, has been confirmed by crystallographic research (Brejc et al. 1997, Palm et al. 1997). The phenol in Tyr⁶⁶ is uncharged in GFP variants with excitation maxima at ~395 nm (corresponding to state A), while it is in the charged phenolate form in variants with excitation maxima at 473 nm (corresponding to state B). Kneen et al. (1998) suggested that pH shifts the equilibrium between the GFP A and B ground states. At high pH, the phenolate form of Tyr⁶⁶ is favored so that the B state is populated (excitation at 471 nm, emission at 500 nm). At low pH, the phenol form is favored so that the state A is populated and the absorbance shifts to 390 nm. The absence of fluorescence at 500 nm by excitation at 360 nm could be due to quenching of the I state or inability of the I state to convert to the B state. The authors noted that GFP pH sensitivity could involve simple protonation events at pH>5, but both protonation and conformational changes at lower pH.

As previously mentioned, different GFP variants have different pH sensitivities. Currently, GFP mutants have been developed with pKas ranging from 4.8 (T203I) to 8.0 (YFP-H148G/T203Y) (Table I). Such a range permits researchers to employ specific GFP variants to successfully study processes in either alkaline or acidic environments.

Because of the relative stability of GFP fluorescence over a wide pH range, GFP can be used as a fusion tag to study various processes from subcellular dynamics to protein localization in different pH environments. Rizzuto et al. (1995) first demonstrated the ability of GFP to be targeted to the mitochondria, allowing the visualization of mitochondrial movement in living cells. Recently, Harada et al. (2000) constructed a chimera of EGFP and Wilson's disease gene product ATP7B. Although it is widely believed that ATP7B is located at the Golgi complex, the results indicated that its main localization is in the late endosomes. By combining previous data with the current results, the authors suggested that ATP7B may translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Furthermore, the disturbed incorporation of copper into the late endosomes caused by defective ATP7B may be the main defect in Wilson's disease.

Insights into molecule transport and secretion have also been obtained through the use of GFP fusions. In Dictyostelium discoideum cells, Hanakam et al. (1996) manipulated pH levels while monitoring intracellular translocation of histactophilin II fused to GFP. The results showed that histactophilin II differs from the majority of myristoylated proteins in that it translocates in a pH-dependent manner to the plasma membrane and does not require the myristoyl residue for membrane association. In leaf cells of Nicotiana clevelandii, Boevink et al. (1998) demonstrated for the first time in vivo brefeldin A-induced retrograde transport of Golgi membrane protein to the endoplasmic reticulum. Data also suggested that the leaf Golgi complex acts as a motile system of actin-directed stacks whose function is to pick up products from a relatively stable endoplasmic reticulum system. Wubbolts et al. (1996) fused GFP to the cytoplasmic tail of the class II beta chain of HLA-DR1 to visualize vesicular transport from lysosomal structures to the cell surface in human melanoma Mel JuSo cells. Results indicated that vesicles containing the chimeras did fuse with the plasma membrane and that this transport route did not intersect the earlier endosomal pathway. In one further example, Han et al. (1999) used a proANF/EGFP chimera expressed in rat PC12 pheochromocytoma cells to demonstrate that calcium influx into the cytoplasm rapidly alkalinizes the contents of the peptidergic secretory vesicles. This result suggests for the first time that the physical state of neuropeptides is changed in preparation for release.

The persistence of GFP fluorescence over a broad pH range has also been exploited to examine pH-induced plant pathogen gene expression. Tang et al. (1999) constructed a Tn5 GFPuv-containing transposon derivative, termed mini-Tn5gfp-km, and used this to identify Agrobacterium tumefaciens genes that were upregulated in response to acidic pH. Also using a GFPuv-containing mini Tn5 transposon, Li et al. (1999) determined that a minimal medium of pH 5.5 was the most representative of the growth conditions that A. tumefaciens cells encounter in plant tissues.

A final example of the use of GFP to study processes in various pH environments is an experiment by Matsuyama et al. (2000) in which the authors targeted YFP-H148G to the mitochondrial matrix to examine early events that modulate caspase activation during apoptosis. Results indicated that the mitochondria-mediated alteration of intracellular pH may be an early event that regulates caspase activation in the mitochondrial pathway for apoptosis.

The heightened pH sensitivity of GFP variants has been exploited to probe pH in living cells. Moreover, GFP has several characteristics that render it a useful pH indicator. GFP does not require ester permeation or hydrolysis of small dyes and is easily targetable to intracellular locations without leakage or migration. Furthermore, though some toxic effects of GFP have been noted (Liu et al. 1999), GFP is less detrimental to cells than chemical indicators requiring invasive loading procedures (Kneen et al. 1998, Llopis et al. 1998). Robey et al. (1998) successfully used GFPMut1 (F64L/S65T) to monitor intracellular pH of BS-C-1 (African green monkey kidney) and rabbit renal proximal tube cells in real time. Kneen et al. (1998) used the same mutant (F64L/S65T) to study cytoplasmic and organellar pH levels and pH regulation. Mitochondrial pH was found to be >7.5, but could not be determined accurately because of the much lower pKa (6.0) of the GFP mutant used. In HeLa cells, Llopis et al. (1998) employed a number of mutants (pKas 6.15-7.1) to measure cytosolic, mitochondrial, and Golgi complex pH. The enhanced yellow fluorescent protein variant (S65G/S72A/T203Y/H231L) was found to be suitable for measuring all three subcellular domains, while EGFP was suitable for cytosolic and Golgi pH measurements.

In an effort to create greater pH-sensitive GFP reporters, Miesenböck et al. (1998) altered key amino acids known from crystallographic studies to either be part of the proton-relay network of Tyr⁶⁶ or to change the excitation spectrum when mutated (Ormö et al. 1996, Brejc et al. 1997). Two types of reporters, termed pHluorins, were generated. The first derivative, ratiometric pHluorin, has excitation peaks at 395 nm and 475 nm and undergoes a spectral shift as pH is changed within the physiologically relevant range. The second derivative, ecliptic pHluorin, behaves as a single wavelength probe with a quench in the 475 nm excitation peak with decreasing pH. These pHluorins were used to determine Golgi complex and endosome pH. Additionally, when fused to a vesicle membrane protein, these reporters were able to monitor the release and recycling of synaptic vesicles in hippocampal neurons, as reflected by pH-induced changes in fluorescence.

Since GFP maintains fluorescence over a broad pH range, it can be successfully employed as a molecular reporter in different pH environments. Outside of the pH 6-10 range, however, GFP fluorescence is modified. Spectroscopic and crystallographic data indicate that the protonation state of the chromophore is responsible for GFP pH sensitivity. A number of pH sensitive mutants have been generated, permitting GFP to be utilized as a pH indicator or to monitor exocytic/endocytic events as reflected by pH-induced changes in fluorescence. Current GFP variants have pKas ranging from 4.8 (T203I) to 8.0 (YFP-H148G/T203Y). Thus, researchers are able to employ specific variants to study processes in either alkaline or acidic environments.

This research was supported by Natural Sciences and Research Council grant #600-16-1 to Dr. Francis Choy and a NSERC PGS-B Scholarship to Tessa Campbell.

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Mutant (Common Name)	pKa	Reference(s)
None (wt, BioGreen)	4.5	Tsien 1998
S65T (GFP-S65T)	5.9, 6.0	Kneen et al.1998, Elsliger et al.1999
F64L/S65T (EGFP,GFPMut1,BioST)	5.8, 6.0, 6.15	Haupts et al.1998, Kneen et al.1998, Llopis et al.1998
S65T/H148D	7.8	Elsliger et al.1999
T203I	4.8	Kneen et al.1998
S65G/S72A/T203Y/H231L (EYFPa)	7.1	Llopis et al.1998
S65G/V68L/S72A/T203Y (YFP, EYFP)	7.0	Wachter et al.1998, Elsliger et al.1999
S65G/V68L/S72A/T203Y/E222Q (YFP-E222Q)	7.0	Elsliger et al.1999
S65G/V68L/S72A/H148G/T203Y (YFP-H148G)	8.0	Wachter et al.1998, Elsliger et al.1999
S65G/V68L/S72A/H148Q/T203Y (YFP-H148Q)	7.5	Elsliger et al.1999
K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L (ECFPa)	6.4	Llopis et al.1998
Y66H (BFP, P4, BioBlue)	6.1	Kneen et al.1998

^a The authors use their own naming system with regards to the EYFP and ECFP variants. These differ in mutational composition from Clontech EYFP and ECFP.