Red-shifted Mutant Research Articles

Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been previously identified. Further characterization revealed that mutations N229T and T352M, in particular, bring about unimodal emission with the majority of photons being >600 nm. The red-shifting mutations identified by this high-throughput approach provide strong biochemical evidence for the multiple-emitter mechanism of color determination and point to the importance of a water network in the enzyme binding pocket for altering the emitter ratio. This work provides a broadly applicable mutational data set tying FLuc structure to emission color that contributes to our mechanistic understanding of emission color determination and should facilitate further engineering of improved probes for deep-tissue imaging.

Increasing the brightness of red fluorescent proteins by saturation mutagenesis

Red fluorescent proteins (RFPs) are genetically-encoded fluorophores that are widely used for in vivo imaging. For all applications of RFPs, brighter variants are desired. Previously, we improved the brightness of mRojoA, a red-shifted mutant of the widely-used RFP mCherry, by designing a triple-decker motif of aromatic rings around its chromophore. This yielded the brighter variant mRojo-VHSV, which contains a triple-decker motif consisting of His and Tyr side chains that pack against the chromophore. This improved chromophore packing resulted in an approximately 3-fold brightness increase at physiological pH. However, the His side chain in the triple-decker motif of mRojo-VHSV adopted a perpendicular arrangement to the other two, which may result in a suboptimal packing arrangement. To further improve chromophore packing in mRojo-VHSV, we performed saturation mutagenesis of residues surrounding its triple-decker motif (positions 62, 97, 165, and 199). Using a microplate fluorescence screening assay, a total of 376 colonies were screened for improved brightness. The brightest mutant found, L199M, was expressed and purified, and its spectral properties were characterized in detail. We found that the quantum yield of this variant was improved by two-fold, resulting in a two-fold brightness increase compared to mRojo-VHSV as well as a 5.3-fold increase in brightness compared to mRojoA. The L199M improved variant is the basis for continued engineering with the goal of further improving the spectral properties of this family of RFPs.

Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.

The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

Development of red-shifted mutants derived from luciferase of Brazilian click beetle Pyrearinus termitilluminans.

Luciferase, a bioluminescent protein, has been used as an analytical tool to visualize intracellular phenomena. Luciferase with red light emission is particularly useful for bioluminescence imaging because of its high transmittance in mammalian tissues. However, the luminescence intensity of existing luciferases with their emission over 600 nm is insufficient for imaging studies because of their weak intensities. We developed mutants of Emerald luciferase (Eluc) from Brazilian click beetle (Pyrearinus termitilluminans), which emits the strongest bioluminescence among beetle luciferases. We successively introduced four amino acid mutations into the luciferase based on a predicted structure of Eluc using homology modeling. Results showed that quadruple mutations R214K/H241K/S246H/H347A into the beetle luciferase emit luminescence with emission maximum at 626 nm, 88-nm red-shift from the wild-type luciferase. This mutant luciferase is anticipated for application in in vivo multicolor imaging in living samples.

Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase

It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca(2+)-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.

Variants of DsRed fluorescent protein: Development of a copper sensor

The fluorescence quenching of drFP583 (DsRed) protein by metal ions was investigated. CuSO4 reversibly and pH dependently quenched the red emission at 583 nm of drFP583. The copper binding constant was 15 mM. Following random mutagenesis, blue- and red-shifted mutants of drFP583 were generated, and their metal sensing properties were examined. Mutant gRF possessed properties similar to green fluorescent protein and had a 18 mM copper binding constant. Mutant Rmu162 had an extraordinary red-shifted emission peak at 620 nm. A third mutant, Rmu13, had dual emission peaks at 500 nm and 583 nm and possessed the properties of a copper sensor with a binding constant of 11 mM.

Red-shifted mutants of green fluorescent protein: reversible photoconversions studied by hole-burning and high-resolution spectroscopy

Mutants of green fluorescent protein (GFP) are usually designed to absorb and emit light as “one color” systems, i.e. with a single, photostable conformation of the chromophore. We have studied two red-shifted GFP-mutants (S65T and EYFP) by means of hole-burning and high-resolution optical spectroscopy at low temperature, and compare the data to those previously reported for RS-GFP. We prove that these GFP-mutants are not “one color” systems because they can be reversibly phototransformed from one conformation into another. The results are rationalized in terms of energy-level schemes that are similar to that previously derived by us for wild-type GFP. In these schemes, each mutant can be interconverted by light among at least three conformations that are associated with the protonation-state of the chromophore. The results have relevance for the study of protein–protein interactions by fluorescence resonance energy transfer (FRET), where GFP-mutants of different colors are used as labels in donor–acceptor pairs. Furthermore, we present a detailed mechanism that explains the “on–off” and “blinking” behavior of single GFP-molecules with the proposed energy-level diagrams.

Green fluorescent antibodies: novel in vitro tools.

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.

One- and Two-Photon Excited Fluorescence Lifetimes and Anisotropy Decays of Green Fluorescent Proteins

We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant ∼3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were ∼1.1 ns (main component) and ∼3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 ± 1 ns). The ∼1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional ∼13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at ∼440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore.

Green fluorescent protein as a selectable marker of retrovirally transduced hematopoietic progenitors.

Recombinant retroviruses are most commonly used in hematopoietic stem cell gene therapy trials, but gene transfer efficiency is still inadequate with the present vectors. One approach for overcoming this problem is to develop methods of selecting and enriching the successfully transduced cells. We investigated the feasibility of using the green fluorescent protein (GFP) gene as a selectable marker of hematopoietic cells. When M1 murine leukemia cells were electroporated with GFP expression vectors, a red-shifted mutant (S65T) GFP showed several-fold greater fluorescence than the wild-type GFP and generated readily detectable green light under control of SRalpha or CAG promoter. We then inserted an SRalpha-S65T GFP cassette into the MSCV retrovirus vector and established virus producer cells. Infection of primary murine bone marrow cells resulted in a distinct population with green fluorescence, which was separated by fluorescence-activated cell sorting. The fractionated bright cells gave rise to fluorescent spleen colonies in lethally irradiated mice, while the fluorescence-negative cells yielded only dark colonies. These results indicated that GFP is a faithful marker in gene transfer into hematopoietic progenitor/stem cells, facilitating selection of the transduced cells and tracking of their progeny in vivo.

Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells.

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 microm and 0.5-1.5 microm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems

The potential of the gfp (green fluorescent protein) gene from the jellyfish Aequoria victoria as a visible marker for tracking lactic acid bacteria introduced into complex anaerobic ecosystems was examined. A red-shifted mutant gfp gene was placed downstream of the constitutive Lactococcus lactis P32 promoter and fluorescence resulting from green fluorescent protein (GFP) expression was detected in Escherichia coli and L. lactis. The construct (pKPSPsgfp) was also introduced into Enterococcus faecalis, Streptococcus gordonii and Streptococcus bovis by mobilisation with pVA797. In all cases fluorescence was detectable at the level of individual cells, and isolated colonies. Although oxygen is required for GFP to fluoresce, colonies or cells grown anaerobically were able to fluoresce upon subsequent exposure to air. A combination of selective plating and colony fluorescence allowed a gfp marked strain of E. faecalis to be enumerated after addition to an anaerobic continuous culture of human faecal flora.

The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems

The potential of the gfp (green fluorescent protein) gene from the jellyfish Aequoria victoria as a visible marker for tracking lactic acid bacteria introduced into complex anaerobic ecosystems was examined. A red-shifted mutant gfp gene was placed downstream of the constitutive Lactococcus lactis P32 promoter and fluorescence resulting from green fluorescent protein (GFP) expression was detected in Escherichia coli and L. lactis. The construct (pKPSPsgfp) was also introduced into Enterococcus faecalis, Streptococcus gordonii and Streptococcus bovis by mobilisation with pVA797. In all cases fluorescence was detectable at the level of individual cells, and isolated colonies. Although oxygen is required for GFP to fluoresce, colonies or cells grown anaerobically were able to fluoresce upon subsequent exposure to air. A combination of selective plating and colony fluorescence allowed a gfp marked strain of E. faecalis to be enumerated after addition to an anaerobic continuous culture of human faecal flora.

Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer

Background: Variants of the green fluorescent protein (GFP) with different colors would be very useful for simultaneous comparisons of multiple protein fates, developmental lineages and gene expression levels. The simplest way to shift the emission color of GFP is to substitute histidine or tryptophan for the tyrosine in the chromophore, but such blue-shifted point mutants are only dimly fluorescent. The longest wavelengths previously reported for the excitation and emission peaks of GFP mutants are 488 and 511 nm, respectively.Results Additional substitutions, mainly in residues 145–163, have improved the brightness of the blue-shifted GFP mutants with histidine and tryptophan in place of tyrosine 66. Separate mutations have pushed the excitation and emission peaks of the most red-shifted mutant to 504 and 514 nm, respectively. At least three different colors of GFP mutants can now be cleanly distinguished from each other under the microscope, using appropriate filter sets. A fusion protein consisting of linked blue- and green-fluorescent proteins exhibits fluorescence resonance energy transfer, which is disrupted by proteolytic cleavage of the linker between the two domains.Conclusion Our results demonstrate that the production of more and better GFP variants is possible and worthwhile. The production of such variants facilitates multicolor imaging of differential gene expression, protein localization or cell fate. Fusions between mutants of different colors may be useful substrates for the continuous in situ assay of proteases. Demonstration of energy transfer between GFP variants is an important step towards a general method for monitoring the mutual association of fusion proteins.