Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin

Yan Y. Goh1, Vladimir Frecer1,2, Bow Ho3 and Jeak L. Ding1,4

1 Department of Biological Sciences and 3 Department of Microbiology, National University of Singapore, 14, Science Drive 4, Singapore 117543, Singapore


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Enhanced green fluorescent protein (EGFP) was selected as a signalling scaffold protein for design of a fluorescent biosensor for bacterial endotoxin [or lipopolysaccharide (LPS)]. Virtual mutagenesis was utilized to model EGFP variants containing binding sites for LPS and lipid A (LA), the bioactive component of LPS. Cationic amphipathic sequences of five alternating basic and hydrophobic residues were introduced to ß-sheets located on the surface of EGFP barrel, in the vicinity of the chromophore. Computational methods were employed to predict binding affinity of Escherichia coli LA, to the models of virtual EGFP mutants. DNA mutant constructs of five predicted best binding EGFP variants were expressed in COS-1 cells. The EGFP-mutant proteins exhibited differential expression and variable degrees of fluorescence yield at 508 nm. The EGFP mutants showed a range of LA binding affinities that corresponded to the computational predictions. LPS/LA binding to the mutants caused concentration-dependent fluorescence quenching. The EGFP mutant, G10 bearing LPS/LA amphipathic binding motif in the vicinity of the chromophore (YLSTQ200–204->KLKTK) captured LA with a dissociation constant of 8.5 µm. G10 yielded the highest attenuation of fluorescence intensity in the presence of LPS/LA and demonstrated capability in fluorescence-mediated quantitative detection of LPS in endotoxin-contaminated samples. Thus, the EGFP mutant can form the basis of a novel fluorescent biosensor for bacterial endotoxin.

Keywords: bacterial endotoxin/EGFP mutants/fluorescent biosensor/molecular modelling/quantitative LPS detection


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Bacterial endotoxin [lipopolysaccharide (LPS)] released from Gram-negative bacteria during infection can cause septic shock in humans. Parrillo et al. (Parrillo et al., 1990Go) have reported annual mortalities connected with toxic shock of approximately 100 000 in the US. Currently, the most widely used industrial method of LPS detection for quality assurance of parenteral drugs and medical devices is the Limulus amoebocyte lysate (LAL) test (Morita et al., 1978Go). This test requires the use of amoebocyte lysate from horseshoe crab, which is subjected to seasonal variations in its composition and sensitivity to LPS and relies on the endangered natural source. Therefore, development of alternative, fast and reliable detection methods for LPS in food, cosmetics, pharmaceuticals and healthcare products is crucial and urgent.

Green fluorescent protein (GFP) is a spontaneously fluorescent 27 kDa protein isolated from jellyfish, Aequorea victoria, that does not require post-translational or chemical modification (Cubitt et al., 1995Go). It comprises 238 residues that form a compact 11-stranded ß-barrel, which encloses the fluorescent p-hydroxybenzylideneimidazolinone formed by cyclization of residues Ser65–Gly67 (Ormo et al., 1996Go). The excitation bands of wild-type GFP absorb at 395 and 475 nm while the emission peak maximum is at 508 nm (Cubitt et al., 1995Go). Enhanced GFP (EGFP) (Heim et al., 1995Go; Cormack et al., 1996Go) has been optimized for expression in mammalian cells by introducing more than 190 silent mutations and contains F64L and S65T residue replacements proximal to the chromophore. When excited at 488 nm, EGFP fluoresces 35 times brighter than the wild-type GFP. Relatively minor effects of molecular environment such as weak intermolecular interactions make fluorescence prone to enhancement or attenuation (De Levie, 1997Go). Metals and charged ligands in close proximity to the chromophore are known to quench fluorescence of GFP in a distance- and concentration-dependent fashion (Richmond et al., 2000Go).

GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Abedi et al. (Abedi et al., 1998Go) have adapted GFP as a scaffold for display of conformationally constrained peptides. Mutagenesis and engineering of GFP into chimeric proteins opened new vistas in physiological indicators and biosensors (Tsien, 1998Go). Baird et al. (Baird et al., 1999Go) explored the tolerance of GFP for circular permutations and insertions and concluded that the folding of GFP scaffold is sufficiently robust to permit creation of new fluorescent indicators. Doi and Yanagawa (Doi and Yanagawa, 1999Go) used protein-engineering techniques for molecular design of a biosensor that combines a molecular recognition site with a signal transduction function. They have shown that insertion of a ß-lactamase binding site to a surface loop of GFP resulted in fluorescence changes upon binding of ß-lactamase inhibitor protein. Richmond et al. (Richmond et al., 2000Go) produced a new class of metal sensors by engineering metal-binding sites onto the surface of GFP.

We report here structure-based rational design, molecular genetic engineering and functionality testing of a novel quantitative non-enzymatic fluorescence-based biosensor for detection of LPS or lipid A (LA), the bioactive component of LPS. LPS/LA-binding motif(s) were introduced into the surface of the EGFP scaffold in the vicinity of the chromophore by site-directed mutagenesis of 5 to 10 native residues, which were selected according to predictions from molecular modelling. Previously, we have shown (Frecer et al., 2000aGo) that LPS or LA can interact and bind to short cationic sequences containing symmetrical amphipathic ß-sheet motifs composed of alternating basic (B), hydrophobic (H) and polar (P) residues of general sequences: BHPHB or BHBHB with dissociation constants in the micromolar range (Frecer et al., unpublished data). The strong field of the charged phosphate groups of LPS/LA interacting with the engineered binding motif in the EGFP mutants (EGFPi) was expected to affect the electronic levels of the chromophore and quench its fluorescence depending on the ligand concentration (Figure 1aGo). Such rational approach based on structural considerations and computational predictions of the affinity of designed EGFPi proteins for LPS/LA has undoubted advantage over earlier reports on random mutation, circular permutation or bulk insertion of fragments since it circumvents large scale screening of recombinant libraries. The resulting EGFPi exhibit concentration-dependent attenuation of fluorescence intensities upon LPS/LA binding. This property of the EGFP mutants was employed for determination of LPS concentration in endotoxin-contaminated samples.




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Fig. 1. (a) EGFP biosensor for LPS/LA. The EGFP scaffold carries an engineered ligand-binding site in the vicinity of the chromophore. Binding of the LPS/LA causes quenching of the fluorescence of the EGFP-based biosensor protein, which is proportional to ligand concentration in the sample. (b) Molecular model of EGFPi mutant interacting with LA. The ribbon representation of the barrel-like structure of the EGFPi mutant (G2) shows the three considered sites for engineering of the ligand-binding motif (blue, KVNFKIR162–168; red, NSHNVY146–151; magenta, YLSTQ200–204). The LA moiety in stick representation is docked to the engineered binding motif KGMAK162–166 (red). The side chains of the basic/polar residues of the binding motif extend out of the barrel and form ion-pairs with negatively charged phosphate groups of LA glucosamine disaccharide headgroup (magenta and red). The hydrophobic acyl chains of LA (green) are attached to the EGFPi surface and preserve their ordered parallel arrangement. Hydrogen atoms were omitted in the LA model for better clarity. The chromophore in stick representation (dark blue) positioned in the centre of the EGFPi barrel is located in the vicinity of the engineered motif.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Model building

Molecular models were built with the Biopolymer module of the Insight II program (Molecular Simulations, CA). The model of Escherichia coli LA was adopted from Frecer et al. (Frecer et al., 2000bGo). Variants of EGFPi were derived from the crystal structure of GFP with residues 65–67 replaced by the chromophore: [2-(1-amino-2-hydroxy-propyl)-4-(4-hydroxy-benzilidene)-5-oxo-4,5-dihydro-imidazol-1-yl]-acetaldehyde (Ormo et al., 1996Go; PDB entry code 1EMA) and subsequently refined by molecular mechanics (MM) optimizations using simple simulated annealing protocol. Side chain replacements in selected ß-strands of the EGFP were carried out to introduce LPS/LA-binding motifs containing cationic amphipathic sequences of BHBHB or BHPHB type with cationic/polar residues pointing outside and hydrophobic residues pointing inside the EGFP barrel (Table IGo). Their initial conformations were selected from the side chain rotamer library (Ponder and Richards, 1987Go).


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Table I. Predicted relative binding affinities of designed EGFPi mutants to LA from E.coli
 
MM calculations

Simulations on the EGFPi and LA:EGFPi complexes were carried out with the Discover program (Molecular Simulations) using all-atom representation. Class II consistent force field CFF91 (Maple et al., 1994Go) was employed. Geometry of all molecular models was optimized using conjugate gradient minimization until convergence was reached at the gradient of 0.01 kcal mol-1 Å-1. Except for the five residues of the binding motif and two neighbouring residues, the backbone of the EGFP scaffold was tethered to its crystal structure conformation by a harmonic restraint potential with the force constant of 100 kcal mol-1 Å-2 in all variants during all simulations. The side chains of all residues were relaxed. The dielectric constant was set to 1 during all simulations and effect of the solvent was included via a continuum model of solvation as described below. A formal charge of –1 è was assigned to each phosphate group of LA and +1 è to the ammonium and guanidium groups of Lys and Arg residues, respectively. All atomic charges were taken from the force field library.

Docking of LA to EGFPi

Flexible induced-fit docking methods based on molecular dynamics (MD) simulation (Frecer et al., 2000aGo) have been used for the docking of LA ligand to the EGFPi models allowing for full flexibility of the ligand and the binding motif (Figure 1bGo). The starting position and orientation of the ligand was selected by interactive docking of LA to the binding sequences in the electrostatic and van der Waals energy grid of the receptor until an initial low energy configuration was reached. The LA moiety was directed by its phosphate groups towards the cationic head groups of Lys and Arg residue side chains of the engineered binding motif. MD simulation with a harmonic pulling force was employed to dock the LA molecule. A minor pulling force was set up between the phosphates and ammonium or guanidium groups with a force constant of 10 kcal mol-1 Å-2 and equilibrium separation of 3Å to increase the efficiency of the docking search. No restraints were imposed upon the ligand geometry. The backbone of the EGFPi scaffold (except the binding site) was tethered to its native conformation. The ligand:receptor system was heated from 0 to 300 K over a period of 5 ps and allowed to equilibrate for 5 ps. The MD trajectory integration time was set to 1 fs and the simulation was carried out using the Verlet algorithm. The data collection simulations were carried out for 100 ps, the trajectory was averaged and recorded every 50 steps over 0.05 ps intervals and the non-bonded atom list was updated every 10 steps. During the first 5 ps period, the average potential energy of the system was computed at 300 K. Ligand:receptor complex configurations with potential energy lower than the average were selected during the data collection phase. The docking search for stable complexes produced more than 100 configurations for each considered EGFPi, which were cooled down using a simulated annealing protocol. It included tethering of heavy atoms in their initial high temperature positions followed by three-step energy minimization. A configuration that exhibited the lowest total energy and lowest ligand:receptor interaction energy (negative values) was selected and used for computation of the LA-binding affinity of the particular EGFPi mutant.

Calculation of binding affinity

The affinity of reversible binding of LA to an EGFPi receptor can be estimated from MM calculations on solvated ligand: receptor complex {LA:EGFPi}aq, ligand {LA}aq and receptor {EGFPi}aq assuming the equilibrium {LA}aq + {EGFPi}aq {leftrightarrow} {LA:EGFPi}aq. Gibbs free energy of the ligand:receptor complex formation, {Delta}Gcomp, can be obtained from standard Gibbs free energies of the associating particles at equilibrium:

(1)
We approximate exact thermodynamic values of the Gibbs free energy for larger systems such as EGFPi, by the expression (Frecer et al., 2000aGo):

(2)
where EMM{EGFPi} stands for the MM potential energy of the mutant protein and Gsolv{EGFPi} is its Gibbs free energy of solvation. The complexation Gibbs free energy for the ligand:receptor binding thus takes into account not only interactions in the complex but also the stability of free ligand, free receptor variant and the effect of solvent upon the ligand and receptor association. Comparison between different EGFPi bearing different binding sequences was done via relative changes in the complexation Gibbs free energy: {Delta}{Delta}Gcomp = {Delta}Gcomp{LA:EGFPi} – {Delta}Gcomp{LA:EGFP} using the native EGFP as the reference structure. The evaluation of relative changes is preferable as it is expected to lead to cancellation of errors caused by the approximate nature of the MM method, solvent effect description and neglect of entropic effects. Relative binding affinity (BA) is related to the relative complexation Gibbs free energy as: BA = –{Delta}{Delta}Gcomp.

The relative enthalpic (interaction) component and the solvent effect contribution to the thermodynamic driving force of ligand and receptor association can be evaluated using:

(3)

(4)
where {Delta}HMM{LA:EGFP} and {Delta}Gsolv{LA:EGFP} represent enthalpic and solvent component of binding affinity of LA to the reference receptor—EGFP.

Calculation of solvent effects

Continuum models of solvation have proven useful in biological applications where the description of bulk solvent effects on larger solutes via explicit solvent models is limited by the size or prohibitive simulation times (Tomasi and Persico, 1994Go). The polarizable continuum model (Miertus et al., 1981Go) and its version for biopolymers (Frecer and Miertus, 1992Go) consider the solvent as a homogeneous medium, characterized by macroscopic properties, such as permittivity, polarizability density and molar volume. They employ rigorous treatment of solute–solvent interactions including the electrostatic, dispersion and repulsion terms and also involve the cavitation term that accounts for the creation of a realistic cavity reproducing van der Waals molecular surface of the solute. A dielectric constant of 1 was used for the solute and 78.5 for water. Atomic radii and charges were taken from the CFF91 force field and atomic polarizabilities of Thole (Thole, 1981Go) were used.

Construction of EGFPi with binding site(s) for LA

The pEGFP-1 plasmid (Clontech, CA) was used as the donor template for mutagenesis. The plasmid was digested at NotI and EcoRI sites and cloned into pBS II SK (Stratagene, CA), which was previously linearized at the same restriction site. pBS-EGFP was then subjected to site-directed mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene) with their corresponding designed primers (Operon, CA) shown in Table IIGo. Single or double LA-binding motif(s) were introduced by stepwise mutagenesis into one or two neighbouring ß-strands of the EGFP molecule, respectively. Mutation type and positions were confirmed by DNA sequencing. After mutagenesis on pBS-EGFPi, the mutated vectors were subjected to NotI and EcoRI restriction enzyme digestion followed by subsequent cloning into pCIneo mammalian expression vector (Promega, WI) to give pCIneo-EGFPi. The pCIneo-EGFPi DNA was purified by Wizard Plus SV Minipreps (Promega).


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Table II. Site-directed mutagenesis, amino acid changes in the EGFPi proteins
 
In vitro coupled transcription and translation (TNT)

The pCIneo-EGFPi DNA plasmids were tested for their expression competency using the in vitro rabbit reticulocyte lysate system coupled transcription and translation reaction (TNT) (Promega). Briefly, 25 µl of non-radioactive reaction mixtures were incubated at 30°C for 90 min and 5 µl of each mixture was diluted with 20 µl of SDS–PAGE loading buffer. Out of this mixture, 15 µl was used for protein expression determination of the respective EGFPi.

Transient overexpression of EGFPi in COS-1 cells

COS-1 cells in 25 cm2 culture flasks were transfected using Lipofectamine PLUS reagent (Gibco-BRL, MD). Briefly, 2 µg of DNA was pre-complexed for 15 min with PLUS Reagent in DMEM (Gibco-BRL) and combined with Lipofectamine reagent for 15 min before transfection into each flask of cells containing fresh medium. The DNA–PLUS–Lipofectamine reagent complex was removed after 3 h of incubation at 37°C and the cells were replenished with fresh medium supplemented with 10% fetal bovine serum (Hyclone, UT). The efficiency of the transfectants was observed for green fluorescence under an inverted phase contrast microscope Axiovert 25 (C. Zeiss, Jena, Germany) under a FITC filter. On day 3 post-transfection, the transfected cells were trypsinized and collected by centrifugation at 100 g for 5 min at 4°C. The cell pellet was resuspended with 100 µl of pyrogen-free water (Baxter, NSW, Australia). The cell lysates were prepared by five cycles of freezing at –80°C for 30 min and thawing at 50°C for 10 min with vortexing between each cycle. The cell lysates were centrifuged twice at 14 000 g for 30 min each at 4°C. The aqueous phase was aliquoted and stored at –80°C before assays. The insoluble pellet fractions were directly dissolved in 2x SDS–PAGE loading buffer to yield 50 µg/µl before further analysis. As controls, wild-type pCIneo and pCIneo-EGFP were separately transfected into COS-1 cells and the transfectants were treated in a similar manner.

Protein quantification and determination of expression level of EGFPi

The total protein in the cell lysate was quantified (Bradford, 1976Go). To determine the expression level of the mutant proteins in the lysates and insoluble fractions, western blot analysis was performed using anti-GFP (Invitrogen, CA) as a probe. The blots were visualized using Supersignal West Pico chemiluminescent substrate (Pierce, IL). The relative amount of EGFPi in the bands was quantified densitometrically using Image Master VDS software (Amersham Pharmacia Biotech, Buckinghamshire, UK) compared to pure recombinant GFP, rGFP (Clontech).

Surface plasmon resonance (SPR) analysis of LA binding to EGFPi

Binding of recombinant EGFPi in the COS-1 cell lysate to E.coli LA, f583 (Sigma, MO) was observed with the Biacore 2000 instrument (Biacore, Uppsala, Sweden) using the HPA sensor chip (Biacore). Briefly, LA at 0.5 mg/ml in PBS was immobilized on the HPA chip according to the manufacturer's specification. Five different concentrations of EGFPi mutants in the cell lysate were flown over the monolayer of LA. The SPR binding response units (RU) as a function of time was measured to determine the binding and dissociation kinetics of EGFPi to the immobilized LA. In all experiments, pyrogen-free water was used as the mobile phase at a flow rate of 10 µl/min. To regenerate the HPA chip, 100 mM NaOH was injected at 20 µl/min until the RU returned to the base-line level. Recombinant GFP (Clontech) was injected as the negative control. The dissociation constant of each LA:EGFPi complex was calculated using BiaEvaluation software, version 3.0.

Fluorescence measurements on LA:EGFPi complexes

To assess changes in the fluorescence of the EGFPi after interaction with LPS/LA, the COS-1 cell lysates containing 2 or 4 pmol of the EGFPi were diluted in 100 µl of pyrogen-free water and subjected to a fluorimetric assay at 37°C in the presence of 2 µl of various amounts of LA ranging from 0.1 to 4 ng. LA was pre-warmed at 37°C, vortexed and sonicated for 5 min and vortexed again immediately before addition to the EGFPi lysate. The fluorescence spectra were measured with an LS-50B spectrofluorimeter (Perkin-Elmer). The optimum excitation wavelength of EGFP lies at 488 nm with the corresponding fluorescence emission peak maximum near 508 nm (Cubitt et al., 1995Go). In the recorded emission scans, emission intensities were collected from 370 to 600 nm while the excitation wavelength was fixed at 488 nm. The scanning speed was fixed at 1500 nm/min and the emission wavelength window was set at 10 nm. Changes in the emitted fluorescent light intensity at 508 nm of the LA:EGFPi complexes due to increasing amounts of LA were recorded.

Detection of endotoxin in contaminated liquid samples

Samples:standard solutions of LPS in pyrogen-free water and MilliQ water were tested for LPS with EGFPi G10 lysate. Fluorescence–concentration standard calibration curve for E.coli LPS 055:B5 from BioWhittaker (1.25–100 pg/µl; 16.9–1350 EU/ml) was established. Similar to fluorescence measurements of LA:EGFPi complexes, 10 µl aliquots of the blank, endotoxin standards or samples were added to 92 µl of COS-1 lysate containing 4 pmol of G10 mutant protein. The fluorescence intensities at 508 nm of the LPS:EGFPi complex were measured. LAL kinetic QCL (BioWhittaker, MD) was used to confirm the presence of LPS in the samples. In a separate trial test, contact lens soaking solutions, exposed autoclaved deionized water, tap water and rainwater were also tested for LPS.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Virtual mutagenesis of EGFPi

The EGFPi mutants capable of LPS/LA binding were designed from EGFP scaffold based on several criteria. To achieve distance-dependent quenching of mutants fluorescence by LPS/LA ligands, three possible ß-strands folded in proximity to phenyl ring of the chromophore were selected as suitable locations for engineering of LPS/LA-binding motif(s). In EGFP, the C{alpha} carbon of the central residue of each of the selected sequence lies within 5–10 Å away from the carbon atom carrying the hydroxyl group of the chromophore phenyl ring, which is oriented towards the side of the ß-barrel containing these sequences. Thus, the following ß-sheet sequences were selected for the binding site construction: NSHNVY146–151, KVNFKIR162–168 and YLSTQ200–204 (Table IGo). Using virtual mutagenesis tools, the sequences were modified to yield 10 different EGFPi models with LPS/LA-binding sequences, which resemble the general minimum binding motifs of LPS/LA BHBHB or BHPHB (Frecer et al., 2000aGo). Two additional EGFPi mutants that comprise two parallel LPS/LA-binding motifs were also modelled. The refined models of the EGFPi (G1–G12) showed that binding motifs in the positions 147–151 and 200–204 display the shortest distances of 5–7 Å to the chromophore. Introduction of basic residues into the binding motif increases the net charge of the EGFPi moiety (QM of EGFP equals to –4 è) and enhances the potency of the mutants to bind anionic LPS or LA.

Prediction of binding affinities of EGFPi to LA

The predicted relative binding affinity in solution (–{Delta}{Delta}Gcomp) of the EGFPi to the anionic LA, estimated by computational methods, showed that the affinity of the cationic binding motifs to the LA moiety is strongly dependent on the actual sequence, its location and neighbouring residues. Figure 2aGo illustrates the engineered sequences of EGFPi: G6, G7, G10, G11 and G12 capable of LA recognition and binding. As an example, Figure 2bGo shows details of molecular structure of the binding motifs of the double-stranded EGFPi mutant G12 and the mode of its interaction with the LA moiety. The affinity of EGFPi to LA increases with increasing number of basic residues in the motif (Table IGo). Thus, the variants G7 and G10, which contain the KXKXK motif (X, any residue with a side chain pointing into the barrel interior) are predicted to bind LA stronger than the reference EGFP ({Delta}{Delta}Gcomp < 0). The double-motif mutants G11 and G12, which comprise five and six Lys residues, respectively, were predicted to possess significantly higher affinity to LA.




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Fig. 2. (a) ß-Barrel scaffold of EGFP with the chromophore and highlighted ß-sheet segments selected for engineering of the LA-binding motifs is shown in ribbon representation. Individual engineered sequences of EGFPi mutants G6, G7, G10, G11 and G12 capable of LA recognition are listed. The anionic phosphates of the LA moiety strongly interact with cationic side chains of lysine in short charged ß-sheet motifs with defined symmetry. (b) Detailed view of the binding motifs of double-stranded EGFPi mutant G12 interacting with LA. Two engineered motifs KHKVK147–151 (red) and KLKTK200–204 (magenta) contain six cationic Lys(+) residues pointing outside of the EGFP ß-barrel wall, which are capable of recognition and binding of the anionic LA moiety. Positively charged ammonium groups (blue) of Lys(+) side chains form ion pairs with negatively charged phosphate groups of the glucosamine disaccharide headgroup of LA. Binding of charged LA molecule to the engineered motifs positioned in the vicinity of the chromophore affects the fluorescence in a manner dependent on the LA concentration. Hydrogen atoms are omitted for better clarity. Colouring scheme of the chromophore: green, carbon; red, oxygen; blue, nitrogen atoms.

 
EGFPi protein expression

The five mutants (G6, G7, G10, G11, G12) with the lowest predicted {Delta}{Delta}Gcomp (highest relative binding affinity) to LA were selected for experimental verification. The in vitro TNT expression of these DNA constructs showed that all were transcribed and translated at comparable levels (Figure 3Go). EGFPi with increasing numbers of Lys substitutions showed increasing pI values (Table IIIGo) and correspondingly reduced levels of protein expression in the COS-1 lysate. Single-motif mutants (G6, G7, G10) were mainly expressed as soluble proteins, whereas the majority of double-motif mutants (G11, G12) were expressed as insoluble proteins.



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Fig. 3. Immunoblotting of TNT and COS-1 cell lysate with EGFPi. (a) No significant difference can be observed in the expression level in the in vitro expression system TNT. (b) In in vivo COS-1 cell expression, the higher the number of lysine substitutions in the EGFP scaffold, the lower is the recombinant protein level detected in the cell lysate. G11 and G12 with five and six Lys replacements, respectively, show extremely low expression. (c) As the number of lysine substitutions increased, the recombinant EGFPi proteins were expressed as insoluble proteins. Based on the intensities of western blot, the representative amount of EGFPi in the COS-1 cell lysate was calculated by comparison with 50 ng of rGFP used as a standard control.

 

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Table III. Characterization of EGFPi mutants
 
EGFPi display different fluorescence intensities

The EGFPi displayed different unit fluorescence intensities at 508 nm when excited at 488 nm (Table IIIGo). Native EGFP (G0) has the highest fluorescence, followed by the G7 mutant. Compared to G0, the mutants G10 and G6 exhibited reduced fluorescence yield of 36 and 28%, while the double-motif mutants, G11 and G12, yielded only 8 and 21% of the G0 fluorescence intensity, respectively.

EGFPi show high binding affinity for E.coli LA

A SPR experiment was employed to determine the binding affinities of EGFPi mutants to LA monolayer. As a control, rGFP (Clontech) and native recombinant EGFP (G0) protein was shown to exhibit no binding to the immobilized LA. The apparent experimental dissociation constants Kd of the five tested EGFPi mutants reflect the relative binding affinity to LA. It is noteworthy that the Kd of the mutant proteins decrease with the increasing number of Lys substitutions in the engineered binding motif(s). Thus, the mutants G11 and G12 exhibit the lowest Kd values. This observation confirms that the number of cationic amino acids and their spatial distribution are important for recognition and binding of LA. These dissociation constants, in low micromolar range (Table IIIGo), correspond to Kd of other LPS/LA-binding peptides and proteins (de Haas et al., 1998Go; Tan et al., 2000Go).

Quenching of fluorescence of EGFPi by LA binding

All three single-motif mutants G6, G7 and G10 showed concentration-dependent quenching of the fluorescence upon addition of LA (Figure 4Go). The fluorescence yield of G11 and G12 was too low to perform similar quenching experiments. The extent of the fluorescence quenching followed almost precisely, the order of predicted binding affinities (–{Delta}{Delta}Gcomp) and LA:EGFPi experimental Kd values. The highest quenching effect was observed for G10, where the greatest decrease in fluorescence intensity was observed at each LA concentration tested, reaching maximum quenching of 28% at the highest amount of 4 ng of LA (Figure 4Go).



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Fig. 4. Relative fluorescence quenching effect for 4 pmol of different EGFPi variants interacting with increasing concentrations of LA. In all data presented, the curves have been normalized with respect to G0. Saturation of the fluorescence quenching is observed at higher LA concentrations that approach the limit of equal molar ratios of LA and EGFPi. The intensity of the fluorescent light emitted by the the COS-1 cell lysate that contained the native EGFP increased proportionally to the concentration of added LA, exhibiting up to 9% enhancement upon addition of 0.1–4 ng LA. On the contrary, all five EGFPi mutants showed fluorescence quenching with increasing amounts of added LA. Similar quenching curves were observed when 2 pmol of EGFPi interacted with 0.1–2 ng of LA. The inset shows emission scan of COS-1 cell lysate, EGFP and G10 mutant with and without LA over 460–560 nm, with excitation at 488 nm: (a) 2 pmol EGFP, (b) 2 pmol EGFP + 500 pg of LA, (c) 4 pmol G10, (d) 4 pmol G10 + 500 pg of LA.

 
EGFPi G10 detects endotoxin in samples

Since EGFPi G10 showed the best quenching effect with LA, endotoxin-contaminated liquid samples from various sources were tested for the presence and concentration of LPS. Results showed that EGFPi G10 is able to detect as low as 50 pg (5 pg/µl; 67.5 EU/ml) of LPS in 10 µl of pyrogen-free water spiked with LPS (Figure 5Go), below which fluorescence quenching was considered non-specific. Thus, 4 pmol of G10 is capable of precise detection of LPS (Table IVGo). Contact lens solutions, tap water, autoclaved water and rainwater caused higher fluorescence quenching, hence showing the presence of more LPS (Table IVGo).



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Fig. 5. Relative fluorescence quenching—LPS concentration standard calibration curves of G10-based detection of LPS. The standard curve of LPS in the optimum detection interval (5–100 pg/µl; 67.5–1350 EU/ml) was constructed by measuring the decrease in fluorescent light intensity of EGFPi G10 relative to EGFP G0 (fluorescence enhanced) after addition of 10 µl of LPS. LPS concentration below this range showed a high degree of noise. Regression analysis of five points of the normalized curve (67.5–1350 EU/ml) furnished a statistically significant equation of Y = 6.87 log X – 10.5, where Y = percentage quenched and X = concentration of endotoxin (r2 = 0.98). In a separate experiment using the LAL (BioWhittaker) test, the lowest detection level of the same LPS is at 0.05 EU/ml.

 

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Table IV. Endotoxin content in samples using EGFPi G10 in COS-1 cell lysate and BioWhittaker LAL test
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Binding affinity of EGFP mutants with LA

G7, G10, G11 and G12 mutants out of 12 considered EGFPi models were predicted to bind LA with higher affinity than the EGFP. EGFP itself contains a native sequence KVNFK162–166, which resembles an LPS/LA-binding motif, but was not predicted to interact strongly with LA. Therefore, it is clear that additional structural and steric factors such as higher positive charge, nature and conformation of residues in the vicinity of the binding motif affect the overall binding affinity of the proteins to the LA moiety. When more Lys residues are substituted into selected ß-sheet sequences of the EGFP molecule (Figure 2aGo) to form a cationic amphipathic pattern of the type BHBHB or BHPHB, the apparent dissociation constants Kd of the EGFPi:LA complexes decreased proportionally. The positively charged ammonium groups of cationic side chains of Lys form ion pairs with the anionic phosphate groups of the glucosamine disaccharide headgroup of the LA moiety and contribute strongly to the LA binding. Such an effect is even more pronounced when two adjacent ß-sheet strands are concurrently substituted to form a dual LPS/LA-binding motif (Figure 2bGo). Thus, increasing number of basic residues in the binding motif (increasing total charge of the EGFP molecule) enhances the interaction of the mutant protein with LA.

Experimental Kd of the LA:EGFPi complexes determined by SPR measurements (Table IIIGo), confirm the dependence of the LA-binding affinity on the number and position of the basic residues and symmetry of the binding motif. The Kd of EGFPi remain in the micromolar and submicromolar range, comparable to that of polymyxin B, a peptidic antibiotic and anti-endotoxin agent (Srimal et al., 1996Go; Thomas and Surolia, 1999Go). The SPR measurements and the resulting apparent Kd were not obtained for purified EGFPi, but only for the COS-1 cell lysate containing the EGFPi recombinant mutants. Even though the reference binding curves of the lysate alone were subtracted we cannot fully exclude the interference of any negatively charged lysate components that could bind to the cationic binding motifs of the EGFPi. Nevertheless, regression analysis of the Kd of five transfected EGFPi constructs that showed the highest predicted affinities to E.coli LA (Table IGo) furnished statistically significant linear correlation with the experimental dissociation constants (Table IIIGo):

where n means number of points, R is the correlation coefficient, F denotes the F-test and {alpha} describes the level of statistical significance. Thus, Kd constants observed by SPR experiment correspond well to LA-binding affinities of the EGFPi predicted from the molecular modelling.

In the SPR experiments, there is no apparent LA binding to either of the wild-type rGFP or EGFP in COS-1 cell lysate, suggesting that the LA-binding activity to the EGFPi should start only at negative {Delta}{Delta}Gcomp values. Reduced predicted binding affinity of G6 compared to G7–G12 was also confirmed by SPR experiment. Bearing in mind the approximate nature of force field-based computational methods, the degree of correlation between experimental results and the modelling predictions is satisfactory.

Differential expression of EGFP mutants

As the number of Lys substitutions in the EGFPi increases, the pI values of the proteins are closer to neutral. Although in vitro TNT expression study displayed similar competency of expression for all mutant constructs, these mutants bearing more Lys residues (double-motif mutants: G11 and G12) were found to be increasingly insoluble in the COS-1 cells. This may be attributed to misfolding of these EGFPi proteins or alternatively, these EGFPi proteins may interact with the anionic phospholipid membrane of COS-1 cells due to the change in their net molecular charge: –4 è for EGFP, –2 è for G6 and +2 è for G12.

LA binding to EGFPi causes differential fluorescence quenching effect

LA binding by the EGFPi mutants, especially by G10, results in significant fluorescence quenching. At 4 ng LA quenched the fluorescence intensity of 4 pmol of G10 by 28%. At higher amounts of LA, the fluorescence quenching curves reach a plateau, as the ratio of LA to EGFPi reaches molar ratio of 1:2. Therefore, G10 may form an excellent molecular model for a fluorescent biosensor for quantitative detection of LPS/LA. Although G11 and G12 mutants displayed the highest affinity for LA (the lowest Kd values), on their own these EGFPi variants exhibited lower fluorescence yields than the G6, G7 and G10, and therefore possess reduced capacity to signal the ligand binding by quenching of the chromophore fluorescence due to low fluorescence yield.

EGFPi G10 as a potential fluorescence sensor for LPS

In the present state, crude EGFPi G10 in the COS-1 cell lysate is capable of detecting endotoxin in standards of known LPS concentrations with considerable precision. With known LPS concentrations in MilliQ water and other liquid samples such as contact lens soaking solutions, tap water, autoclaved water and rainwater, some non-specific interactions of the COS-1 lysate proteins with other components of the liquid samples are anticipated, hence, yielding higher background fluorescence quenching. In order to eliminate possible non-specific interactions and interference of the fluorescence quenching, purified EGFPi mutants with high fluorescence yields and increased specificity to LPS should be tested. To this end, a separate study using stably expressed EGFPi G10 was first tested and found to be able to detect LPS based on its fluorescence quenching. Further purification of EGFPi G10 and optimizations are currently being undertaken in order to reach higher sensitivity. Besides purity of the mutant proteins, optimal buffer or ionic strengths of solvents, the affinity and specificity have to be taken into consideration for further improvement of this fluorescence-based biosensor. Apart from fluorescent intensities, alternative detection methods such as life-time fluorescence decay and single photon excitation upon molecular interaction with LPS could also be considered.

It is noteworthy that mutation N149K in both single and double-motif mutants increased the fluorescence yield by ~3-fold. It also decreased the apparent Kd of the LA:EGFPi complexes by ~5-fold. In addition, binding of LA induced a similar degree of fluorescence quenching in the mutants containing the N149K substitution. Thus, replacement of the single polar Asn149 with charged Lys residue seems to improve both the fluorescence yield, binding of LA and the quenching effect on the chromophore, and is a subject of current research in this laboratory.

Furthermore, virtual mutagenesis modelling of additional amino acid substitutions, T62F or T62Y in the EGFPi G11 and G12, indicates that the rigidity of the chromophore is increased by stacking interactions between the aromatic rings of Phe62 or Tyr62 and the phenyl ring of the chromophore, which may lead to enhanced fluorescence yields of these mutants with the highest affinities to the LA. The EGFPi G11 and G12 variants that include T62Y replacement would be preferable since they have the potential to detect lower LPS concentrations due to lower dissociation constants than the G10. However, this hypothesis remains to be verified experimentally.


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
The experimental results confirmed that the folding-robust EGFP scaffold with its native fluorescence, prone to quenching after ligand binding can be used for rational design of a biosensor for detection of LPS/LA. Computer-aided molecular modelling and virtual mutagenesis enabled the design of specific mutations, which engineered an LPS/LA-binding motif(s) into the vicinity of the chromophore. Concentration-dependent fluorescence quenching by the LPS/LA ligands is suitable for quantitative detection of the ligand concentration. A rational design strategy circumvents the need for expensive and laborious random mutation and screening processes of combinatorial mutant libraries before any selection and evaluation can be performed. Although the LPS/LA binding to EGFPi G10 may not be the strongest, this mutant represents a promising candidate for further biosensor design due to its high fluorescence intensity and high quenching effect upon LPS/LA interaction. If the initial fluorescence yield can be further enhanced by additional residue replacements, EGFPi G12 will predictably form the most sensitive LPS/LA receptor.


    Notes
 
2 Present address: Cancer Research Institute, Slovak Academy of Sciences, SK-83391 Bratislava, Slovakia Back

4 To whom correspondence should be addressed. E-mail: dbsdjl{at}nus.edu.sg Back


    Acknowledgments
 
This work was supported by the National Science and Technology Board (NSTB grant no. LS/99/004). Goh Y.Y. is a research scholar of the National University of Singapore.


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Received May 4, 2001; revised November 14, 2001; accepted December 7, 2001.