Green fluorescent protein variants as ratiometric dual emission pH sensors. 3. Temperature dependence of proton transfer

In parts 1 and 2 of this series [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S. Y., Xi, L. X., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488; McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], we described the structure, excited-state dynamics, and applications of pH-sensitive, ratiometric dual emission green fluorescent protein (deGFP) variants with fluorescence emission that is modulated between blue (lambda(max) approximately equal 465 nm) and green (lambda(max) approximately equal 515 nm) depending on the pH of the bulk solvent. In this paper, we consider the energetic origin of the dual emission properties of these GFP variants by examining the temperature dependence of the steady-state absorption and fluorescence emission. In most cases, the quantum yield of the green emission decreased as the temperature was lowered, indicating that the excited-state proton transfer (ESPT) which produces the green emitting form is an activated process. The activation energies of ESPT, determined by modeling the quantum yields of both blue and green emissions between 260 and 298 K in the context of a simple photocycle, were found to be larger at low pH than at high pH. These results indicate that the ratiometric dual emission properties of deGFP mutants are due to this pH-sensitive ESPT rate, combined with a modulation of the ground-state neutral and anionic chromophore populations with pH. The time-resolved fluorescence of one of the deGFP mutants was studied in detail. The time-resolved emission spectra of this mutant are the first ultrafast spectra obtained for a GFP. These spectra demonstrate that the rising kinetics for green emission, considered a hallmark of ESPT, is the sum of the contribution from both the neutral and intermediate anionic forms of the chromophore at the probe wavelength and may not be observed in all mutants that undergo ESPT, depending on the relative contributions of the two forms.     

Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application

Novel dual emission, pH-sensitive variants of the green fluorescent protein (GFP) have been constructed and are suitable for ratiometric emission measurements in vivo. This new class of GFPs, termed deGPFs, results from substitution of wild-type residue 65 with threonine and residues 148 and/or 203 with cysteine. deGFPs display pK(a) values ranging from 6.8 to 8.0 and emission that switches from a green form (lambda(max) approximately 515 nm) to a blue form (lambda(max) approximately 460 nm) with acidifying pH. In this report we analyze in most detail the deGFP1 variant (S65T/H148G/T203C, pK(a) approximately 8.0) and the deGFP4 variant (S65T/C48S/H148C/T203C, pK(a) approximately 7.3). In the following paper [McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], data obtained by ultrafast fluorescence upconversion spectroscopy can be described by a kinetic model that includes an excited-state proton-transfer pathway at high pH but not at low pH. Crystal structure analyses of deGFP1 at high-pH and low-pH conformations were performed to elucidate the basis for the dual emission characteristics. At low pH the structure does not contain a hydrogen bond network that would support rapid transfer of a proton from the excited state of the neutral chromophore to a suitable acceptor; hence blue emission is observed. At high pH, backbone rearrangements induced by changes in the associated hydrogen bond network permit excited-state proton transfer from the excited state of the neutral chromophore to the bulk solvent via Ser147 and bound water molecules, resulting in green emission from the anionic chromophore. Comparative analysis suggests that the basis for dual emission is elimination of the wild-type proton-transfer network by the S65T substitution, a general reduction in hydrogen-bonding opportunities, and a concomitant increase in the hydrophobic nature of the chromophore environment resulting from the cysteine substitutions. We evaluated the suitability of the deGFP4 variant for intracellular pH measurements in mammalian cells by transient expression in PS120 fibroblasts. The responses of deGFP4 and a commercially available pH-sensitive dye, SNARF-1, to changes in pH were compared in the same cells. Results show that the dynamic range of the emission ratio change is comparable between the two pH sensors over the range examined. Two-photon excitation was found to elicit a better deGFP4 fluorescent signal above cellular autofluorescence when compared to conventional confocal microscopy. Given their favorable optical characteristics, suitable pK(a)'s for the physiological pH range, and suitability for ratiometric measurements, dual emission GFPs should make excellent probes for studying pH in vivo.     

Green fluorescent protein as a noninvasive intracellular pH indicator.

It was found that the absorbance and fluorescence of green fluorescent protein (GFP) mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (GFP-F64L/S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T203I) with apparent Hill coefficients of 0.7 for Y66H and approximately 1 for the other proteins. For GFP-S65T in aqueous solution in the pH range 5-8, the fluorescence spectral shape, lifetime (2.8 ns), and circular dichroic spectra were pH independent, and fluorescence responded reversibly to a pH change in <1 ms. At lower pH, the fluorescence response was slowed and not completely reversed. These findings suggest that GFP pH sensitivity involves simple protonation events at a pH of >5, but both protonation and conformational changes at lower pH. To evaluate GFP as an intracellular pH indicator, CHO and LLC-PK1 cells were transfected with cDNAs that targeted GFP-F64L/S65T to cytoplasm, mitochondria, Golgi, and endoplasmic reticulum. Calibration procedures were developed to determine the pH dependence of intracellular GFP fluorescence utilizing ionophore combinations (nigericin and CCCP) or digitonin. The pH sensitivity of GFP-F64L/S65T in cytoplasm and organelles was similar to that of purified GFP-F64L/S65T in saline. NH4Cl pulse experiments indicated that intracellular GFP fluorescence responds very rapidly to a pH change. Applications of intracellular GFP were demonstrated, including cytoplasmic and organellar pH measurement, pH regulation, and response of mitochondrial pH to protonophores. The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH.     

Synthesis and photophysical properties of new SNARF derivatives as dual emission pH sensors

We report the synthesis and properties of two new seminaphthorhodafluor (SNARF) derivatives, SNARF-F and SNARF-Cl. Both these derivatives exhibit typical red shifts of absorption and fluorescence, and higher cell permeability as compared to traditional SNARF, while the pH-dependent dual-emission characteristics are well retained. In particular, the lower pK_a (7.38) of SNARF-F makes it more suitable than traditional SNARF derivatives for intracellular applications.     

Green fluorescent protein with tryptophan-based chromophore stable at low pH

Using directed (substitution T203Y) and subsequent random mutagenesis of the monomeric cyan fluorescent protein mTurquoise2, we obtained a protein with a tryptophan-based chromophore that fluoresces in the green region of the spectrum (excitation maximum 482 nm, emission maximum 519 nm). Fluorescence of the new protein is highly stable in a wide range of pH (pK _a 4.9), more stable than all monomeric green fluorescent proteins with a tyrosine-based chromophore.     

Green fluorescent protein as a marker for Pseudomonas spp.

The development of sensitive methods for observing individual bacterial cells in a population in experimental models and natural environments, such as in biofilms or on plant roots, is of great importance for studying these systems. We report the construction of plasmids which constitutively express a bright mutant of the green fluorescent protein of the jellyfish Aequorea victoria and are stably maintained in Pseudomonas spp. We demonstrate the utility of these plasmids to detect individual cells in two experimental laboratory systems: (i) the examination of a mixed bacterial population of Pseudomonas aeruginosa and Burkholderia cepacia attached to an abiotic surface and (ii) the association of Pseudomonas fluorescens WCS365 with tomato seedling roots. We also show that two plasmids, pSMC2 and pGB5, are particularly useful, because they are stable in the absence of antibiotic selection, they place an undetectable metabolic burden on cells that carry the plasmids, and cells carrying these constructs continue to fluoresce even after 7 days in culture without the addition of fresh nutrients. The construction of improved Escherichia coli-Pseudomonas shuttle vectors which carry multiple drug resistance markers also is described.     

Green fluorescent protein as a signal for protein-protein interactions.

Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was chosen as a model system. GFP-tagged S-peptide was produced, and the interaction of this fusion protein with S-protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein-protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein-protein interactions.     

Targeted optical probing of neuronal circuit dynamics using fluorescent protein sensors

Interest in non-invasive methods for optical probing of neuronal electrical activity has been ongoing for several decades and methods for imaging the activity of single or multiple individual neurons in networks composed of thousands of neurons have been developed. Most widely used are techniques that use organic chemistry-based dyes as indicators of calcium and membrane potential. More recently a new generation of probes, genetically encoded fluorescent protein sensors, have emerged for use by physiologists studying the operation of neuronal circuits. In this review we describe the advance of these emerging optical techniques and compare them with more conventional approaches.     

Engineering green fluorescent protein as a dual functional tag

A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.     

Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains.

To measure pH in defined intracellular microdomains of living cells, we developed ratiometric indicators based on fusing in tandem two green fluorescent protein (GFP) variants having different pH sensitivities. The indicators function in a single-excitation/dual-emission mode involving fluorescence resonance energy transfer, as well as in a dual-excitation/single-emission mode. The fluorescence ratio from GFpH and YFpH showed pH dependency and pK(a) values were 6.1 and 6.8, respectively. Using these indicators expressed in cultured cells, we measured and visualized pH changes in the cytosol and nucleus. Furthermore, by tethering the indicator to a membrane protein (the alpha(1B) adrenergic receptor), we visualized the pH in the vicinity of the protein during internalization caused by endocytosis after agonist stimulation. These novel probes will serve as a useful tool for monitoring pH in the defined organelle and in the microenvironment of a target protein, to analyze cellular function.     

Gramicidin derivatives as membrane-based pH sensors.

Ion channels provide a means for sensitive pH measurement at membrane interfaces. Detailed knowledge of the structure and function of gramicidin channels permits the engineering of pH-sensitive derivatives. Two derivatives, gramicidin-ethylenediamine and gramicidin-histamine, are shown to exhibit pH-dependent single-channel behaviour over the pH ranges 9-11 and 6.5-8.5, respectively. Thermal isomerization of a carbamate group at the entrance of the channels leads to a pattern of steps in single-channel recordings. The size of the steps depends on the time-averaged degree of protonation of the appended group (ethylenediamine or histamine). Measurement of the size of the steps thus permits single-molecule pH sensing under symmetrical pH conditions or in the presence of a pH gradient.     

Structural and spectral response of green fluorescent protein variants to changes in pH

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mutants of GFP respond rapidly and reversibly to pH variations, making them useful as probes of intracellular pH. To explore the structural basis for the titration behavior of the popular GFP S65T variant, we determined high-resolution crystal structures at pH 8.0 and 4.6. The structures revealed changes in the hydrogen bond pattern with the chromophore, suggesting that the pH sensitivity derives from protonation of the chromophore phenolate. Mutations were designed in yellow fluorescent protein (S65G/V68L/S72A/T203Y) to change the solvent accessibility (H148G) and to modify polar groups (H148Q, E222Q) near the chromophore. pH titrations of these variants indicate that the chromophore pKa can be modulated over a broad range from 6 to 8, allowing for pH determination from pH 5 to pH 9. Finally, mutagenesis was used to raise the pKa from 6.0 (S65T) to 7.8 (S65T/H148D). Unlike other variants, S65T/H148D exhibits two pH-dependent excitation peaks for green fluorescence with a clean isosbestic point. This raises the interesting possibility of using fluorescence at this isosbestic point as an internal reference. Practical real time in vivo applications in cell and developmental biology are proposed.     

Spectral and photophysical studies of benzo[c]xanthene dyes: dual emission pH sensors

A series of fluorescent, long-wavelength, benzo[c]-xanthene dyes has been characterized for pH measurement in both excitation and emission ratio applications. The two general classes of these indicators are seminaphthofluoresceins (SNAFLs) and seminaphthorhodafluors (SNARFs) which are substituted at the 10-position with oxygen or nitrogen, respectively. These probes show separate emissions from the protonated and deprotonated forms of the fluorophores. The dyes may be excited at 488 or 514 nm with argon ion lasers. Most of the indicators have pKa values between 7.6 and 7.9. Detailed photophysical studies were conducted on the carboxy-SNAFL-1 system and excited-state prototropic reactions were compared to structurally related derivatives, such as the umbelliferones. Membrane permeant esters, such as diacetates and acetoxymethyl esters have also been prepared. The indicators are spectrally well resolved from calcium indicators such as fura-2 and indo-1 and should be suitable for simultaneous determination of pH and Ca2+ transients.     

Green fluorescent protein as a scaffold for intracellular presentation of peptides.

Peptide aptamers provide probes for biological processes and adjuncts for development of novel pharmaceutical molecules. Such aptamers are analogous to compounds derived from combinatorial chemical libraries which have specific binding or inhibitory activities. Much as it is generally difficult to determine the composition of combinatorial chemical libraries in a quantitative manner, determining the quality and characteristics of peptide libraries displayed in vivo is problematical. To help address these issues we have adapted green fluorescent protein (GFP) as a scaffold for display of conformationally constrained peptides. The GFP-peptide libraries permit analysis of library diversity and expression levels in cells and allow enrichment of the libraries for sequences with predetermined characteristics, such as high expression of correctly folded protein, by selection for high fluorescence.     

Green fluorescent protein as an all-purpose reporter in Petunia

Two critical attributes of a reporter gene are ease of scoring for activity and capacity for expression in all cell types. We have examined a variant of the gene encoding green fluorescent protein,mgfp5, for its ability to meet these criteria in petunia. Under regulation of the Cauliflower Mosaic Virus (CaMV) 35S promoter, GFP was detectable in all vegetative and most floral cell types. Promoters from petuniaadhl andadh2 allowed for production of GFP in those few cell types lacking GFP production from the CaMV 35S promoter, verifying its capacity for expression in all cell types. With the appropriate promoter, GFP fluorescence was thus readily detectable throughout the plant. A potential complication is the green autofluorescence exhibited by some plant tissues. This auto-fluorescence is for the most part distinguishable from that contributed by GFP, but under-scores the need for appropriate controls in GFP-reporter-based experiments. An erratum to this article is available at .     

Green fluorescent protein as a marker in transgenic mice

Green fluorescent protein (GFP) found in Aequorea victoria absorbs blue light and emits green fluorescence without exogenous substrates or co-factors. We studied the possibility of using the GFP as a marker in mammals. Transgenic mice were produced using the GFP coding sequence, ligated with the chicken beta-actin promoter. Green fluorescence was observed in muscle, pancreas, kidney, heart and other organs in all the three transgenic mouse lines. Detection of the transgenic mouse was possible by observing a tail or fingers of new born pups under a fluorescent microscope. The marker also enabled us to detect localized expression of the transgene in intact tissues without preliminary steps. It was also demonstrated that the GFP expression could be quantified by measuring the fluorescence in tissue extracts.     

Green fluorescent protein as a molecular marker in microbiology

Molecular markers such as: lacZ (b-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (beta-glucuronidase), gfp (green fluorescent protein), bla (beta-lactamase) and other antibiotic resistance markers, heavy metals resistance genes are commonly used in environmental microorganisms research (Errampaii et al., 1998; Kohler et al., 1999). Most of these markers require one or more substrates, complex media and/or expensive equipment for detection. The gfp gene is widely used as a marker because of its very useful properties such as high stability, minimal toxicity, non-invasive detection and the ability to generate the green light without addition of external cofactors and without application of expensive equipment. Various applications of that reporter gene were showed starting from monitoring of microorganism's survival in complex biological systems such as activated sludge to biodegradation of chemical compounds in soil. GFP allowed the detection, determination of spatial location and enumeration of bacterial cells from diverse environmental samples such as biofilm and water. The gfp as a biomarker was very useful in monitoring of gene expression and protein localisation in bacterial cells, too. The techniques with using gfp marker promise to supply a better understanding of environmental processes. It can make possible to use that knowledge in designing more effective and more efficient methods of biodegradation of toxic compounds from different environments.     

Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli.

Escherichia coli is one of the most widely used vehicles to overexpress membrane proteins (MPs). Currently, it is not possible to predict if an overexpressed MP will end up in the cytoplasmic membrane or in inclusion bodies. Overexpression of MPs in the cytoplasmic membrane is strongly favoured to overexpression in inclusion bodies, since it is relatively easy to isolate MPs from membranes and usually impossible to isolate them from inclusion bodies. Here we show that green fluorescent protein (GFP), when fused to an overexpressed MP, can be used as an indicator to monitor membrane insertion versus inclusion body formation of overexpressed MPs in E. coli. Furthermore, we show that an overexpressed MP can be recovered from a MP-GFP fusion using a site specific protease. This makes GFP an excellent tool for large-scale MP target selection in structural genomics projects.