Rational design of analyte channels of the green fluorescent protein for biosensor applications

A novel solvent-exposed analyte channel, generated by F165G substitution, on the surface of green fluorescent protein (designated His(6)GFPuv/F165G) was successfully discovered by the aid of molecular modeling software (PyMOL) in conjunction with site-directed mutagenesis. Regarding the high predictive performance of PyMOL, two pore-containing mutants namely His(6)GFPuv/H148G and His(6)GFPuv/H148G/F165G were also revealed. The pore sizes of F165G, H148G, and the double mutant H148G/F165G were in the order of 4, 4.5 and 5.5 A, respectively. These mutants were subjected to further investigation on the effect of small analytes (e.g. metal ions and hydrogen peroxide) as elucidated by fluorescence quenching experiments. Results revealed that the F165G mutant exhibited the highest metal sensitivity at physiological pH. Meanwhile, the other 2 mutants lacking histidine at position 148 had lower sensitivity against Zn(2+) and Cu(2+) than those of the template protein (His(6)GFPuv). Hence, a significant role of this histidine residue in mediating metal transfer toward the GFP chromophore was proposed and evidently demonstrated by testing in acidic condition. Results revealed that at pH 6.5 the order of metal sensitivity was found to be inverted whereby the H148G/F165G became the most sensitive mutant. The dissociation constants (K(d)) to metal ions were in the order of 4.88 x 10(-6) M, 16.67 x 10(-6) M, 25 x 10(-6) M, and 33.33 x 10(-6) M for His(6)GFPuv/F165G, His(6)GFPuv, His(6)GFPuv/H148G/F165G and His(6)GFPuv/H148G, respectively. Sensitivity against hydrogen peroxide was in the order of H148G/F165G > H148G > F165G indicating the crucial role of pore diameters. However, it should be mentioned that H148G substitution caused a markedly decrease in pH- and thermo-stability. Taken together, our findings rendered the novel pore of GFP as formed by F165G substitution to be a high impact channel without adversely affecting the intrinsic fluorescent properties. This opens up a great potential of using F165G mutant in enhancing the sensitivity of GFP in future development of biosensors.     

The conserved His-144 in the PsbP protein is important for the interaction between the PsbP N-terminus and the Cyt b559 subunit of photosystem II

The PsbP protein regulates the binding properties of Ca(2+) and Cl(-), and stabilizes the Mn cluster of photosystem II (PSII); however, the binding site and topology in PSII have yet to be clarified. Here we report that the structure around His-144 and Asp-165 in PsbP, which is suggested to be a metal binding site, has a crucial role for the functional interaction between PsbP and PSII. The mutated PsbP-H144A protein exhibits reduced ability to retain Cl(-) anions in PSII, whereas the D165V mutation does not affect PsbP function. Interestingly, H144A/D165V double mutation suppresses the effect of H144A mutation, suggesting that these residues have a role other than metal binding. FTIR difference spectroscopy suggests that H144A/D165V restores proper interaction with PSII and induces the conformational change around the Mn cluster during the S(1)/S(2) transition. Cross-linking experiments show that the H144A mutation affects the direct interaction between PsbP and the Cyt b(559) α subunit of PSII (the PsbE protein). However, this interaction is restored in the H144A/D165V mutant. In the PsbP structure, His-144 and Asp-165 form a salt bridge. H144A mutation is likely to disrupt this bridge and liberate Asp-165, inhibiting the proper PsbP-PSII interaction. Finally, mass spectrometric analysis has identified the cross-linked sites of PsbP and PsbE as Ala-1 and Glu-57, respectively. Therefore His-144, in the C-terminal domain of PsbP, plays a crucial role in maintaining proper N terminus interaction. These data provide important information about the binding characteristics of PsbP in green plant PSII.     

Co-expression of zinc binding motif and GFP as a cellular indicator of metal ions mobility

A significant role of zinc-binding motifs on metal mobility in Escherichia coli was explored using a chimeric metal-binding green fluorescent protein (GFP) as an intracellular zinc indicator. Investigation was initiated by co-transformation and co-expression of two chimeric genes encoding the chimeric GFP carrying hexahistidine (His6GFP) and the zinc-binding motif fused to outer membrane protein A (OmpA) in E. coli strain TG1. The presence of these two genes was confirmed by restriction endonucleases analysis. Co-expression of the two recombinant proteins exhibited cellular fluorescence activity and enhanced metal-binding capability of the engineered cells. Incorporation of the zinc-binding motif onto the membrane resulted in 60-fold more binding capability to zinc ions than those of the control cells. The high affinity to metal ions of the bacterial surface influenced influx of metal ions to the cells. This may affect the essential ions for triggering important cell metabolism. A declining of fluorescent intensity of GFP has been detected on the cell expressed of zinc binding motif. Meanwhile, balancing of metal homeostasis due to the presence of cytoplasmic chimeric His6GFP enhanced the fluorescent emission. These findings provide the first evidence of real-time monitoring of intracellular mobility of zinc by autofluorescent proteins.     

Structure-function analysis of Friedreich's ataxia mutants reveals determinants of frataxin binding and activation of the Fe-S assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k(cat)/K(M) higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k(cat)/K(M) of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.     

Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids

Bacterial allantoinase (ALLase) and dihydroorotase (DHOase) are members of the cyclic amidohydrolase family. ALLase and DHOase possess similar binuclear metal centers in the active site in which two metals are bridged by a post-translationally carboxylated lysine. In this study, we determined the effects of carboxylated lysine and metal binding on the activities of ALLase and DHOase. Although DHOase is a metalloenzyme, purified DHOase showed high activity without additional metal supplementation in a reaction mixture or bacterial culture. However, unlike DHOase, ALLase had no activity unless some specific metal ions were added to the reaction mixture or culture. Substituting the metal binding sites H59, H61, K146, H186, H242, or D315 with alanine completely abolished the activity of ALLase. However, the K146C, K146D and K146E mutants of ALLase were still active with about 1–6 % activity of the wild-type enzyme. These ALLase K146 mutants were found to have 1.4–1.7 mol metal per mole enzyme subunit, which may indicate that they still contained the binuclear metal center in the active site. The activity of the K146A mutant of the ALLase and the K103A mutant of DHOase can be chemically rescued by short-chain carboxylic acids, such as acetic, propionic, and butyric acids, but not by ethanol, propan-1-ol, and imidazole, in the presence of Co²⁺ or Mn²⁺ ions. However, the activity was still ~10-fold less than that of wild-type ALLase. Overall, these results indicated that the 20 natural basic amino acid residues were not sufficiently able to play the role of lysine. Accordingly, we proposed that during evolution, the post-translational modification of carboxylated lysine in the cyclic amidohydrolase family was selected for promoting binuclear metal center self-assembly and increasing the nucleophilicity of the hydroxide at the active site for enzyme catalysis. This kind of chemical rescue combined with site-directed mutagenesis may also be used to identify a binuclear metal center in the active site for other metalloenzymes.     

Divalent Metal Ion-Induced Folding Mechanism of RNase H1 from Extreme Halophilic Archaeon Halobacterium sp. NRC-1

RNase H1 from Halobacterium sp. NRC-1 (Halo-RNase H1) is characterized by the abundance of acidic residues on the surface, including bi/quad-aspartate site residues. Halo-RNase H1 exists in partially folded (I) and native (N) states in low-salt and high-salt conditions respectively. Its folding is also induced by divalent metal ions. To understand this unique folding mechanism of Halo-RNase H1, the active site mutant (2A-RNase H1), the bi/quad-aspartate site mutant (6A-RNase H1), and the mutant at both sites (8A-RNase H1) were constructed. The far-UV CD spectra of these mutants suggest that 2A-RNase H1 mainly exists in the I state, 6A-RNase H1 exists both in the I and N states, and 8A-RNase H1 mainly exists in the N state in a low salt-condition. These results suggest that folding of Halo-RNase H1 is induced by binding of divalent metal ions to the bi/quad-aspartate site. To examine whether metal-induced folding is unique to Halo-RNase H1, RNase H2 from the same organism (Halo-RNase H2) was overproduced and purified. Halo-RNase H2 exists in the I and N states in low-salt and high-salt conditions respectively, as does Halo-RNase H1. However, this protein exists in the I state even in the presence of divalent metal ions. Halo-RNase H2 exhibits junction ribonuclease activity only in a high-salt condition. A tertiary model of this protein suggests that this protein does not have a quad-aspartate site. We propose that folding of Halo-RNase H1 is induced by binding of divalent metal ion to the quad-aspartate site in a low-salt condition.     

Structural and thermodynamic analysis of the GFP:GFP-nanobody complex

The green fluorescent protein (GFP)-nanobody is a single-chain VHH antibody domain developed with specific binding activity against GFP and is emerging as a powerful tool for isolation and cellular engineering of fluorescent protein fusions in many different fields of biological research. Using X-ray crystallography and isothermal titration calorimetry, we determine the molecular details of GFP:GFP-nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding. Although the GFP-nanobody can also bind YFP, it cannot bind the closely related CFP or other fluorescent proteins from the mFruit series. CFP differs from GFP only within the central chromophore and at one surface amino acid position, which lies in the binding interface. Using this information, we have engineered a CFP variant (I146N) that is also able to bind the GFP-nanobody with high affinity, thus extending the toolbox of genetically encoded fluorescent probes that can be isolated using the GFP-nanobody.     

DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu(+) and Cu(2+) ions

The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu(+) and Cu(2+), with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The K(d) values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu(2+) (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling.     

Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy

Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development.     

Site-directed mutagenesis in the B-neuropilin-2 domain selectively enhances its affinity to VEGF165, but not to semaphorin 3F

Neuropilins (NRPs) are 130-kDa receptors that bind and respond to the class 3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF165 and SEMA3F. Cell binding studies reveal that VEGF165 and SEMA3F bind NRP2 with similar affinities, 5.2 and 3.9 nM, respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF165 binding, whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF165 binding, point mutations were introduced by site-directed mutagenesis. VEGF165 is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A,E291A) in the 280-290 b1-NRP2 loop resulted in a 2-fold reduction in VEGF165 affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E,N290D and R287E,N290S) significantly increased VEGF165 affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF165 affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF165 binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF165/NRP2 interactions.     

Thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the urease metallochaperone UreE

The two Ni2+ ions in the urease active site are delivered by the metallochaperone UreE, whose metal binding properties are central to the assembly of this metallocenter. Isothermal titration calorimetry (ITC) has been used to quantify the stoichiometry, affinity, and thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the well-studied C-terminal truncated H144*UreE from Klebsiella aerogenes, Ni2+ binding to the wild-type K. aerogenes UreE protein, and Ni2+ and Zn2+ binding to the wild-type UreE protein from Bacillus pasteurii. The stoichiometries and affinities obtained by ITC are in good agreement with previous equilibrium dialysis results, after differences in pH and buffer competition are considered, but the concentration of H144*UreE was found to have a significant effect on metal binding stoichiometry. While two metal ions bind to the H144*UreE dimer at concentrations <10 microM, three Ni2+ or Cu2+ ions bind to 25 microM dimeric protein with ITC data indicating sequential formation of Ni/Cu(H144*UreE)4 and then (Ni/Cu)2(H144*UreE)4, or Ni/Cu(H144*UreE)2, followed by the binding of four additional metal ions per tetramer, or two per dimer. The thermodynamics indicate that the latter two metal ions bind at sites corresponding to the two binding sites observed at lower protein concentrations. Ni2+ binding to UreE from K. aerogenes is an enthalpically favored process but an entropically driven process for the B. pasteurii protein, indicating chemically different Ni2+ coordination to the two proteins. A relatively small negative value of DeltaCp is associated with Ni2+ and Cu2+ binding to H144*UreE at low protein concentrations, consistent with binding to surface sites and small changes in the protein structure.     

The metal binding site of zoocin A

Direct metal analysis of the bacteriolytic exoenzyme zoocin A failed to unequivocally identify a putative metal cofactor; hence, indirect experiments utilizing NMR were undertaken to settle this question. Cd(2+) as a surrogate metal ion was reconstituted into EDTA-treated, metal-free recombinant zoocin, and (113)Cd-NMR was employed to explore binding in the protein for this ion. The Cd-substituted enzyme was found to have 80-85% of native streptococcolytic activity. A major (113)Cd resonance at 113.6 ppm was observed which with time split into resonances at 113.6 and 107.2 ppm. A minor (113)Cd resonance at 87.3 ppm was observed which increased in intensity with time. These Cd chemical shifts are indicative of two N atoms and two O atoms ligating directly to the metal site.On the basis of conserved amino acid residues in a homologous protein of known structure, LytM, the ligands in zoocin are tentatively assigned to H45, D49, H133, and some combination of water or buffer ions as the fourth oxygen donor in zoocin A. Comparison of the combined intensities for (113)Cd-substituted zoocin with a known quantity of another Cd-substituted protein gave Cd binding as approximately stoichiometric (1.2 +/- 0.2) with protein. Additional metal-removal and reconstitution experiments on the recombinant catalytic domain of zoocin implicate Zn(2+) as the metal cofactor. Therefore, the evidence supports zoocin as a single Zn(2+) ion binding metalloenzyme.     

Effect of divalent metal ions and glycerol on the GTPase activity of H-ras proteins

The product of the protooncogenic ras gene (p21N ras) exhibits a weak GTPase activity. A significant increase in the GTPase activity associated with p21N ras protein was obtained by using glycerol in the assay mixture. Of the several metal ions tested, only Mg++ and Mn++ are effective divalent cations that support the GTPase activity of p21N ras protein. p21N ras protein exhibits higher GTPase activity and yields higher [3H] GDP binding in the presence of MnCl2 than with MgCl2. Optimal GTPase and [3H] GDP binding are obtained at micromolar concentrations of MgCl2 or MnCl2. Concentrations in the millimolar range of either MgCl2 or MnCl2 are inhibitory to the GTPase activity, whereas [3H] GDP binding was not affected.     

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.     

Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solid-supported lipid membrane by quartz crystal microbalance and atomic force microscopy

Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn2+, was formed on the gold electrode of quartz resonator (5 MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device.     

Metal ion binding and enzymatic mechanism of Methanococcus jannaschii RNase HII

RNase H degrades the RNA moiety in DNA:RNA hybrid in a divalent metal ion dependent manner. It is essential to understand the role of metal ion in enzymatic mechanism. One of the key points in this study is how many metal ions are involved in the enzyme catalysis. Accordingly, either one-metal binding mechanism or two-metal binding mechanism is proposed. We have studied the thermodynamic properties of four metal ions (Mg(2+), Mn(2+), Ca(2+), and Ba(2+)) binding to Methanococcus jannaschii RNase HII using isothermal titration calorimetry. All of the four metal ions were found to bind Mj RNase HII with 1:1 stoichiometry in the absence of substrate. Together with enzymatic activity assay data, we propose that only one metal ion binding to the enzyme in catalytic process. We also studied the pH dependence of metal binding and enzyme activity and found that at pH 6.5, Mg(2+) did not bind to the enzyme without the substrate but still activated the enzyme to about 2% of its maximum activity (in 10 mM Mn(2+) at pH 8). This implies that the substrate may also be incorporated in metal ion binding and help to position the metal ion. To find which acidic residues correspond to metal ion binding, we also studied the binding thermodynamics and enzymatic activity assay of four mutants: D7N, E8Q, D112N, and D149N in the presence of Mn(2+). The thermodynamic parameters are least affected for the D149N mutant, which has a very low enzymatic activity. This indicates that Asp149 is essential for the enzymatic activity. On the basis of all these observations, we suggest a metal binding model in which D7, E8, and D112 bind the metal ion and D149 activates a water molecule to attack the P-O bond in the RNA chain of the substrate.     

Metal ion complexes of apoferritin. Evidence for initial binding in the hydrophilic channels

Metal binding to the iron storage protein apoferritin is the first step in the process by which iron accumulates within the protein shell. In the present study, the stoichiometry of metal binding to apoferritin in solution has been examined using the probe ions Mn(II), VO(IV), and Cd(II) in conjunction with EPR spectroscopic and cadmium ion selective electrode measurements. Binding studies were carried out with the individual ions, in competition with one another, and in competition with Fe(II), Fe(III), and Tb(III). All three probe ions show binding stoichiometries near 0.3 and 0.7 metal ion per subunit, close to the theoretically predicted values of 0.33 and 0.67 for the binding of one and two metal ions, respectively, per three subunits. These results in conjunction with other data are consistent with the binding of one, and possibly two, metal ions within each of the eight hydrophilic channels which are located on 3-fold axes leading to the interior of the protein. Pairs of cadmium binding sites have been located in these channels by x-ray crystallography (Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A., and Harrison, P. M. (1983) Adv. Inorg. Biochem. 5, 39-49). The possibility that some metal binding occurs elsewhere on the protein is not precluded by the present data, however. In competition experiments between various metal ions, approximately 0.3 metal ion per subunit is readily displaced implying common binding sites in the channels for all of them. The stoichiometry of Mn(II) displacement by Fe(II) is less clear. Oxidation of Fe(II) to Fe(III) by molecular oxygen in the presence of Mn(II) regenerates some Mn(II) binding on the protein, suggesting migration of iron(III) to other protein sites, or perhaps to core.     

Facilitating chromophore formation of engineered Ca binding green fluorescent proteins

Green fluorescent protein (GFP) containing a self-coded chromophore has been applied in protein trafficking and folding, gene expression, and as sensors in living cells. While the “cycle3” mutation denoted as C3 mutation (F99S/M153T/V163A) offers the ability to increase GFP fluorescence at 37 °C, it is not clear whether such mutations will also be able to assist the folding and formation of the chromophore upon the addition of metal ion binding sites. Here, we investigate in both bacterial and mammalian systems, the effect of C2 (M153T/V163A) and C3 (F99S/M153T/V163A) mutations on the folding of enhanced GFP (EGFP, includes F64L/S65T) and its variants engineered with two types of Ca²⁺ binding sites: (1) a designed discontinuous Ca²⁺ binding site and (2) a grafted continuous Ca²⁺ binding motif. We show that, for the constructed EGFP variants, the C2 mutation is sufficient to facilitate the production of fluorescence in both bacterial and mammalian cells. Further addition of the mutation F99S decreases the folding efficiency of these variants although a similar effect is not detectable for EGFP, likely due to the already greatly enhanced mutation F64L/S65T from the original GFP, which hastens the chromophore formation. The extinction coefficient and quantum yield of purified proteins of each construct were also examined to compare the effects of both C2 and C3 mutations on protein spectroscopic properties. Our quantitative analyses of the effect of C2 and C3 mutations on the folding and formation of GFP chromophore that undergoes different folding trajectories in bacterial versus mammalian cells provide insights into the development of fluorescent protein-based analytical sensors.     

Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand

The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.