Interaction of Genome-linked Protein (VPg) of Turnip Mosaic Virus with Wheat Germ Translation Initiation Factors eIFiso4E and eIFiso4F
2006; Elsevier BV; Volume: 281; Issue: 38 Linguagem: Inglês
10.1074/jbc.m605479200
ISSN1083-351X
AutoresMateen A. Khan, Hiroshi Miyoshi, Sibnath Ray, Tomohide Natsuaki, Noriko Suehiro, Dixie J. Goss,
Tópico(s)Viral Infections and Immunology Research
ResumoThe interaction between VPg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIFiso4E for VPg. Scatchard analysis revealed the binding affinity (Ka) and average binding sites (n) for VPg were (8.51 ± 0.21) × 106 M–1 and 1.0, respectively. The addition of eIFiso4G to the eIFiso4E increased the binding affinity 1.5-fold for VPg as compared with eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy-favorable with a large negative ΔH°(–29.32 ± 0.13 kJmol–1) and positive ΔS° (36.88 ± 0.25 Jmol–1K–1). A Lineweaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-7-methylguanosine triphosphate for eIFiso4E. Fluorescence stopped-flow studies of eIFiso4E and eIFiso4F with VPg show rapid binding, suggesting kinetic competition between VPg and m7G cap. The VPg protein binds much faster than cap analogs. The activation energies for binding of eIFiso4E and eIFiso4F with VPg were 50.70 ± 1.27 and 75.37 ± 2.95 kJmol–1 respectively. Enhancement of eIFiso4F-VPg binding with the addition of a structured RNA derived from tobacco etch virus suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome. The interaction between VPg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIFiso4E for VPg. Scatchard analysis revealed the binding affinity (Ka) and average binding sites (n) for VPg were (8.51 ± 0.21) × 106 M–1 and 1.0, respectively. The addition of eIFiso4G to the eIFiso4E increased the binding affinity 1.5-fold for VPg as compared with eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy-favorable with a large negative ΔH°(–29.32 ± 0.13 kJmol–1) and positive ΔS° (36.88 ± 0.25 Jmol–1K–1). A Lineweaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-7-methylguanosine triphosphate for eIFiso4E. Fluorescence stopped-flow studies of eIFiso4E and eIFiso4F with VPg show rapid binding, suggesting kinetic competition between VPg and m7G cap. The VPg protein binds much faster than cap analogs. The activation energies for binding of eIFiso4E and eIFiso4F with VPg were 50.70 ± 1.27 and 75.37 ± 2.95 kJmol–1 respectively. Enhancement of eIFiso4F-VPg binding with the addition of a structured RNA derived from tobacco etch virus suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome. Viral RNAs share characteristics with the host cell mRNAs but must employ different strategies for preferential translation. Recently attention has focused on the possible role of the viral protein linked to the genome (VPg) 2The abbreviations used are: VPg, viral protein linked to the genome; TuMV, turnip mosaic virus; eIF, eukaryotic initiation factor; IRES, internal ribosomal entry sites; Ant, anthraniloyl; m7GTP, 7-methylguanosine triphosphate; DTT, dithiothreitol; TEV, tobacco etch virus; PK1, Pseudoknot 1; S1-3, stem mutation on PK1 of TEV.2The abbreviations used are: VPg, viral protein linked to the genome; TuMV, turnip mosaic virus; eIF, eukaryotic initiation factor; IRES, internal ribosomal entry sites; Ant, anthraniloyl; m7GTP, 7-methylguanosine triphosphate; DTT, dithiothreitol; TEV, tobacco etch virus; PK1, Pseudoknot 1; S1-3, stem mutation on PK1 of TEV. (1Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Laliberte J.F. Roberts L. EMBO Rep. 2005; 6: 968-972Crossref PubMed Scopus (156) Google Scholar, 2Leonard S. Plante D. Wittmann S. Daigneault N. Fortin M.G. 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Virol. 1989; 70: 2785-2789Crossref PubMed Scopus (55) Google Scholar) covalently linked to the 5′-end. The potyviral VPg is multifunctional, and interaction between VPg and eIF4E has been reported for TuMV (2Leonard S. Plante D. Wittmann S. Daigneault N. Fortin M.G. Laliberte J.-F. J. Virol. 2000; 74: 7730-7737Crossref PubMed Scopus (238) Google Scholar, 8Wittmann S. Chatel H. Fortin M.G. Laliberte J.F. Virology. 1997; 234: 84-92Crossref PubMed Scopus (214) Google Scholar), but the consequences of this interaction with respect to translation initiation are not clear because elements in the 5′-untranslated region of the potyviral RNA direct cap-independent translation (9Carrington J. Freed D. J. Virol. 1990; 64: 1590-1597Crossref PubMed Google Scholar). Viral proteins are likely to participate in the regulation of viral genome translation (10Thompson S.R. Sarnow P. Curr. Opin. Microbiol. 2000; 3: 366-370Crossref PubMed Scopus (42) Google Scholar, 11Gale Jr., M. Tan S.L. Katze M.G. Microbiol. Mol. Biol. Rev. 2000; 64: 239-280Crossref PubMed Google Scholar). VPg has several suggested roles in the virus life cycle. Interactions of VPg with the viral RNA polymerase in yeast (12Hong Y. Levay K. Murphy J.F. Klein P.G. Shaw J.G. Hunt A.G. Virology. 1995; 214: 159-166Crossref PubMed Scopus (77) Google Scholar, 13Li X.H. Valdez P. Olvera R.E. Carrington J.C. J. Virol. 1997; 71: 1598-1607Crossref PubMed Google Scholar) and in vitro (14Fellers J. Wan J. Hong Y. Collins G.B. Hunt A.G. J. Gen. Virol. 1998; 79: 2043-2049Crossref PubMed Scopus (60) Google Scholar) support a role in viral RNA synthesis. Additionally, VPg has been implicated in overcoming resistance in plants (15Keller K.E. Johansen I.E. Martin R.R. Hampton R.O. Mol. Plant-Microbe Interact. 1998; 11: 124-130Crossref PubMed Scopus (106) Google Scholar, 16Masuta C. Nishimura M. Morishita H. Hataya T. Phytopathology. 1999; 89: 118-123Crossref PubMed Scopus (68) Google Scholar, 17Nicolas O. Dunnington S.W. Gotow L.F. Pirone T.P. Hellmann G.M. Virology. 1997; 237: 452-459Crossref PubMed Scopus (111) Google Scholar, 18Nicolas O. Pirone T.P. Hellmann G.M. Arch. Virol. 1996; 141: 1535-1552Crossref PubMed Scopus (17) Google Scholar, 19Schaad M.C. Lellis A.D. Carrington J.C. J. Virol. 1997; 71: 8624-8631Crossref PubMed Google Scholar). VPg-Pro of tobacco etch potyvirus also interacts with eIF4E from tomato and tobacco, and the interaction was shown to enhance genome amplification (20Schaad M.C. Anderberg R.J. Carrington J.C. Virology. 2000; 273: 300-306Crossref PubMed Scopus (125) Google Scholar).In wheat germ (Triticum aestivum) and other plants, an isoform of eIF4F called eIFiso4F has been found (21Browning K.S. Lax S.R. Ravel J.M. J. Biol. Chem. 1987; 262: 11228-11232Abstract Full Text PDF PubMed Google Scholar, 22Browning K.S. Webster C. Roberts J.K. Ravel J.M. J. Biol. Chem. 1992; 267: 10096-10100Abstract Full Text PDF PubMed Google Scholar). eIFiso4F functions like eIF4F in supporting cap-dependent in vitro translation (22Browning K.S. Webster C. Roberts J.K. Ravel J.M. J. Biol. Chem. 1992; 267: 10096-10100Abstract Full Text PDF PubMed Google Scholar). However, eIF4F and eIFiso4F show preferences for translation of structured and unstructured non-coding regions, respectively. eIFiso4F contains two subunits, a 28-kDa eIFiso4E and an 86-kDa eIFiso4G. The eIFiso4E acts as the binding site of m7GpppN cap. eIFiso4G binds to mRNA in an ATP-dependent manner, may interact with eIF4A and eIF4B (23Balasta M.L. Carberry S.E. Friedland D.E. Perez R.A. Goss D.J. J. Biol. Chem. 1993; 268: 18599-18603Abstract Full Text PDF PubMed Google Scholar), and more interestingly, may interact with poly(A)-binding protein (24Le H. Tanguay R.L. Balasta M.L. Wei C.C. Browning K.S. Metz A.M. Goss D.J. Gallie D.R. J. Biol. Chem. 1997; 272: 16247-16255Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 25Wei C.C. Balasta M.L. Ren J. Goss D.J. Biochemistry. 1998; 37: 1910-1916Crossref PubMed Scopus (112) Google Scholar). Wheat germ eIF4F consists of only two subunits, a 26-kDa eIF4E and a 220-kDa eIF4G in a 1:1 molar ratio. Some structural and functional similarity exists between eIF4F and eIFiso4F. Functionally, they are very similar even though these two proteins are antigenically distinct (26Lax S. Fritz W. Browning K. Ravel J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 330-333Crossref PubMed Scopus (60) Google Scholar). Wheat germ eIFiso4F can substitute for mammalian eIF4F in an RNA-dependent ATPase activity and in cross-linking of mammalian eIF4A to the cap of oxidized mRNA (27Abramson R.D. Browning K.S. Dever T.E. Lawson T.G. Thach R.E. Ravel J.M. Merrick W.C. J. Biol. Chem. 1988; 263: 5462-5467Abstract Full Text PDF PubMed Google Scholar).The interaction between the VPg of TuMV and the translation eukaryotic initiation factor eIFiso4E and eIFiso4F of Arabidopsis thaliana has previously been reported (2Leonard S. Plante D. Wittmann S. Daigneault N. Fortin M.G. Laliberte J.-F. J. Virol. 2000; 74: 7730-7737Crossref PubMed Scopus (238) Google Scholar, 8Wittmann S. Chatel H. Fortin M.G. Laliberte J.F. Virology. 1997; 234: 84-92Crossref PubMed Scopus (214) Google Scholar). These observations suggest VPg is important in initiation of protein synthesis, perhaps functioning as a cap analogue, as proposed by Herbert and co-workers (28Herbert T.P. Brierley I. Brown T.D. J. Gen. Virol. 1997; 78: 1033-1040Crossref PubMed Scopus (114) Google Scholar). Michon et al. (29Michon T. Estevez Y. Walter J. German-Retana S. Le Gall O. FEBS J. 2006; 273: 1312-1322Crossref PubMed Scopus (80) Google Scholar) characterized the interaction of lettuce eIF4E and VPg from lettuce mosaic virus. Earlier studies (30Miyoshi H. Suehiro N. Tomoo K. Muto S. Takahashi T. Tsukamoto T. Ohmori T. Natsuaki T. Biochimie (Paris). 2006; 88: 329-340Crossref PubMed Scopus (58) Google Scholar) have shown that interaction between VPg and plant eIFiso4E is sufficiently strong so that VPg competes with cap binding. In this study we further characterize this interaction, determine the effects of eIFiso4G on the VPg and eIFiso4E interaction, and demonstrate an interaction with viral RNA. The kinetics of binding suggest that VPg binding to eIFiso4F is favored. We propose a mechanism where VPg substitutes for the cap analog and enhances formation of an eIFiso4F complex with viral internal ribosomal entry sites (IRES).EXPERIMENTAL PROCEDURESMaterials—The TuMV full-length cDNA clone and construction of the expression vector for TuMV VPg was described previously (30Miyoshi H. Suehiro N. Tomoo K. Muto S. Takahashi T. Tsukamoto T. Ohmori T. Natsuaki T. Biochimie (Paris). 2006; 88: 329-340Crossref PubMed Scopus (58) Google Scholar, 31Suehiro N. Natsuaki T. Watanabe T. Okuda S. J. Gen. Virol. 2004; 85: 2087-2098Crossref PubMed Scopus (93) Google Scholar). Glutathione-Sepharose 4B and m7GTP-Sepharose were purchased from Amersham Biosciences. Nickel-nitrilotriacetic acid Superflow was purchased from Qiagen K. K. The HiTrap Mono Q, HiTrap SP column, and PreScission protease were purchased from Amersham Biosciences. The pET21a and pET28a expression vectors were purchased from Novagen, an affiliate of Merck. Buffer A was 20 mm Tris-HCl, pH 8.0, 300 mm NaCl, 10 mm imidazole. Buffer B was 20 mm Tris-HCl, pH 8.0, 300 mm NaCl, 250 mm imidazole. Buffer C was 20 mm Tris-HCl, pH 8.0, 10 mm NaCl, 1 mm DTT, and 5% glycerol. Buffer D was 20 mm Tris-HCl, pH 7.6, 150 mm NaCl, and 5% glycerol.Expression and Purification of Recombinant Proteins—eIFiso4E and eIFiso4G were expressed in Escherichia coli containing the constructed pET3d vector in BL21 (DE3) pLysS as described elsewhere (32van Heerden A. Browning K.S. J. Biol. Chem. 1994; 269: 17454-17457Abstract Full Text PDF PubMed Google Scholar). A HiTrap Mono Q ion exchange column and an m7-GTP-Sepharose column were used for the purification of eIFiso4E. E. coli cells were disrupted by sonication, suspended in buffer B-600 (20 mm HEPES/KOH, pH 7.6, 1 mm DTT, 0.1 mm EDTA, 5% glycerol, 600 mm KCl) containing 0.5 mm phenylmethylsulfonyl fluoride (PMSF), 0.5 ml of aprotinin, and 100 μg/ml soybean trypsin inhibitor. The lysed cells were centrifuged at 15,000 rpm in a Sorvall SS-34 rotor for 30 min (S11 supernatant) to separate soluble eIFiso4E from inclusion bodies. The S11 supernatant was centrifuged at 45,000 rpm in a Sorvall TV-850 rotor for 3 h (S175 supernatant) to remove ribosomes and additional aggregates. The S175 supernatant was dialyzed against buffer B-50 (20 mm HEPES/KOH, pH 7.0, 5% glycerol, 1 mm DTT, 0.1 mm EDTA, 50 mm KCl). The dialyzed sample was applied to a 5-ml HiTrap Mono Q column equilibrated with 10 bed volumes of buffer B-50 with a flow rate of 2 ml/min. The column was washed at the same flow rate with buffer B-50 until optical density returned to base line. The expressed eIFiso4E was eluted with 50–400 mm KCl linear gradient at a flow rate of 2 ml/min. The peak was collected in 1.0-ml fractions. To maximize purity, the sample was subsequently applied to a 4-ml m7GTP-Sepharose column equilibrated in buffer B-50. The column was washed with buffer B-50 followed by 25 ml of buffer B-50 containing 0.1 mm GTP to remove GTP-binding proteins. The expressed eIFiso4E was eluted from the column with 15 ml of buffer B-50 containing 100 mm GTP. After 2 ml of the elution buffer had entered the column, the column was turned off for 30 min. Elution was resumed, and column fractions of 0.5 ml were collected. The fractions were analyzed by 12.5% SDS-polyacrylamide gel electrophoresis. A HiTrap SP column was used to purify eIFiso4G by the following procedure (32van Heerden A. Browning K.S. J. Biol. Chem. 1994; 269: 17454-17457Abstract Full Text PDF PubMed Google Scholar). E. coli cells were disrupted by alumina, suspended in buffer B-600, and centrifuged at 45,000 rpm for 2 h. The supernatant was dialyzed against buffer B-50, loaded onto a 5-ml HiTrap SP column, and washed with B-50 buffer until the optical density returned to the base line. A 50–400 mm KCl linear gradient (total volume 100 ml) was used to elute the eIFiso4G, and 1.0-ml fractions were collected. The eIFiso4G appeared in the 200–300 mm KCl fractions. After purification, the purity was confirmed by 10% SDS-polyacrylamide gel electrophoresis. All steps were carried out in a cold box at ∼5 °C.For purification of TuMV VPg (30Miyoshi H. Suehiro N. Tomoo K. Muto S. Takahashi T. Tsukamoto T. Ohmori T. Natsuaki T. Biochimie (Paris). 2006; 88: 329-340Crossref PubMed Scopus (58) Google Scholar) E. coli BL21(DE3)pLysS cells were transformed with pETVPg1. Cells were cultured in LB medium containing 100 μg/ml ampicillin and 20 μg/ml chloramphenicol at 25 °C. At an A600 of 0.5, expression was induced for 6 h with 50 μm isopropyl 1-thio-β-d-galactopyranoside, after which cells were harvested by centrifugation. Subsequent steps were performed at 4 °C. Cell pellets were resuspended into buffer A and disrupted by sonication, and the lysates were centrifuged. Supernatant was applied to a 1-ml nickel-nitrilotriacetic acid Superflow column equilibrated with buffer A. The column was washed 3 times with 10 ml of buffer A, and the bound protein was eluted with 5 ml of buffer B. The protein was dialyzed against buffer C, further purified to homogeneity on Mono Q and HiLoad 16/60 Superdex 75 pg columns, and finally dialyzed against buffer D. The purified VPg-His6 was confirmed by 15% SDS-PAGE.All samples were dialyzed against buffer B (20 mm HEPES/KOH, pH 7.6, 100 mm KCl, 1.0 mm MgCl2, 1.0 mm dithiothreitol, 1.0 mm EDTA) and passed through a 0.22 μm filter (Millipore) before the spectroscopy measurements were performed. The fractions were concentrated with a Centricon 10 (Amicon Co.) as necessary. The concentrations of protein were determined by a Bradford assay with bovine serum albumin as standard (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar) using a Bio-Rad protein assay reagent (Bio-Rad).The Tobacco Etch Virus (TEV) mRNA Synthesis in Vitro—The (TEV PK1 and S1-3 of PK1) clones were described previously (34Zeenko V. Gallie D.R. J. Biol. Chem. 2005; 280: 26813-26824Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). DNA was linearized with SalI, a site immediately upstream of the luc open reading frame. The linearized DNA was treated with 50 mm Tris-HCl, pH 7.5, containing proteinase K (100 μg/ml), 0.5% SDS, and 5 mm CaCl2 for 30 min at 37 °C. DNA was further purified by phenol:chloroform:isoamyl alcohol (25:24:1) at pH 8.0 and ethanol precipitation. Purity was confirmed by 1% agarose gel electrophoresis, and concentration was determined spectrophotometrically after linearization and brought to 0.5 mg/ml. In vitro transcription of PK1 and S1-3 of PK1 were carried out using Promega RiboMAX™ large scale RNA Production System-T7. Setting of reaction conditions and purification of synthesized RNA was according to the protocol as described in Promega RiboMAX™ kit. The concentration of RNA was determined by measuring the optical density at 260 nm. The purity of synthesized RNA was checked by measuring the absorbance ratio A260/A280 nm in diethyl pyrocarbonate-treated water.Fluorescence Titration Measurements—Fluorescence measurements were performed using a Spex Fluorolog τ2 spectrofluorimeter equipped with excitation and emission polarizers. The formation of the binary protein-protein and protein-RNA complexes was studied by direct fluorescence titration. The excitation and emission slits were set on 4 and 5 nm, respectively. The excitation wavelength for eIFiso4E was 280 nm, and emission was monitored at 332 nm. The excitation slits were chosen to avoid photobleaching, and the absorbance of the sample at the excitation wavelength was less than 0.02 to minimize the inner-filter effect. Emission spectra were corrected for the wavelength-dependent lamp intensity and monochromator sensitivities.The samples were thermostated at the different temperatures, i.e. 5, 10, 15, 22, 25, and 32 ± 0.5 °C. The temperature was monitored by a thermocouple inside the cuvette. Titrations were performed in 20 mm HEPES/KOH, pH 7.6, 100 mm KCl, 1.0 mm MgCl2, and 1.0 mm DTT. The normalized fluorescence difference (ΔF/ΔFmax) between the protein-protein complex and the sum of the individual fluorescence spectra was used to determine the equilibrium dissociation constant (Kd). The details of the data fitting are described elsewhere (35Khan M.A. Goss D.J. Biochemistry. 2004; 43: 9092-9097Crossref PubMed Scopus (23) Google Scholar). Fluorescence intensities, when necessary, were corrected for dilution and for inner filter effect. Nonlinear least squares fitting of the data were performed using KaleidaGraph software (Version 2.1.3; Abelbeck Software).Determination of the Number of Binding Sites—Protein-protein interaction of genome-linked protein (VPg) of turnip mosaic virus with wheat germ eIFiso4E and eIFiso4F (complex of eIFiso4E and eIFiso4G) were studied by direct fluorescence titration. A solution of purified eIFiso4E (0.5 μm) was titrated with increasing amounts of VPg protein. The fluorescence intensity at emission maximum (332 nm) was used for calculating the relative fluorescence, considering the fluorescence intensity of control untreated eIFiso4E as 100. The fractional quench, Q, was determined at each VPg/eIFiso4E molar ratio (R). For an observed fluorescence intensity F, the fractional quench, Q, was obtained from the equation Q = (Fo – F)/m (maximal quench)). Fractional quench, Q, is linearly related to VPg binding, [VPg-eIFiso4E]/[eIFiso4E]T = Q, where [eIFiso4E]T represents the total protein concentration. The average number of binding sites (n) were determined from intercept on the x axis of the Scatchard plot Q versus Q/(R – Q)[eIFiso4E]T (36Khan M.A. Muzammil S. Musarrat J. Int. J. Biol. Macromol. 2002; 30: 243-249Crossref PubMed Scopus (116) Google Scholar, 37Levine R.L. Clin. Chem. 1977; 23: 2292-2301Crossref PubMed Scopus (95) Google Scholar). The slope also gives Ka, which is in agreement with Ka obtained from nonlinear least square fitting.Thermodynamic Parameters for eIFiso4E and eIFiso4F Binding to VPg—The temperature dependence of Keq for VPg was analyzed according to the van't Hoff isobaric equation assuming the entropy change, ΔS°, and the enthalpy change, ΔH°, as constants over the range of temperatures studied.-lnKeq=1RΔHoT-ΔSoCompetitive Binding Experiments—The fluorescent cap analogue, anthraniloyl (Ant)-m7GTP, was synthesized as described previously (38Ren J. Goss D.J. Nucleic Acids Res. 1996; 24: 3629-3634Crossref PubMed Scopus (30) Google Scholar). The competitive substitution reactions were performed at constant Ant-m7GTP concentration (0.1 μm) and increasing amounts of VPg. The fluorescence measurements were made at 25 °C in 20 mm HEPES/KOH, pH 7.6, 100 mm KCl, 1.0 mm MgCl2, and 1.0 mm DTT. An excitation wavelength of 332 nm was used to monitor the Ant-m7GTP fluorescence emission at 420 nm.Stopped-flow Fluorescence Kinetics—Stopped-flow fluorescence experiments were performed on an OLIS RSM 1000 stopped-flow system with a 1-ms dead time. The excitation wavelength was 280 nm, and the cut-on filter was 324 nm for eIFiso4E and eIFiso4F with VPg interactions. A reference photomultiplier was used to monitor fluctuations in the lamp intensity. The temperature of the flow cell and solution reservoirs was maintained using a temperature-controlled circulating water bath. VPg binding induced a decrease in eIFiso4E fluorescence. After rapid mixing of 1 μm (0.5 μm after mixing) eIFiso4E or eIFiso4F with 5 μm (2.5 μm after mixing) VPg, the time course of the fluorescence intensity change was recorded by computer data acquisition. In each experiment, 1000 pairs of data were recorded, and sets of data from 3 experiments were averaged. Each averaged set of stopped-flow data was then fitted to nonlinear analytical equations using Global analysis software provided by OLIS. Data were fitted to the single and double exponential functions. Fitted curves correspond to the single exponential equation (39Olsen K. Christensen U. Sierks M.R. Svensson B. Biochemistry. 1993; 32: 9686-9693Crossref PubMed Scopus (54) Google Scholar),Ft=ΔFexp(-kobst)+F∞ where Ft is that fluorescence observed at any time, t, ΔF is the amplitude, ΔF∞ is the final value of fluorescence, and kobs is the observed first-order rate constant. The kinetic data for double exponential fitsFt=ΔF1exp(-kobs1t)+ΔF2exp(-kobs2t)+F∞ where ΔF1 and ΔF2 are the amplitudes of two exponentials with rate constants kobs1 and kobs2, respectively. The residuals were measured by the differences between the calculated fit and the experimental data. The reaction was consistent with a single exponential process. The derived rate constants were used to construct an Arrhenius plot according to the equation,lnk=-EaRT+lnA(Eq. 4) where k is the rate constant, Ea is the activation energy, and A is the Arrhenius pre-exponential term. The activation energy was calculated from the slope of the fitted linear plot of ln k versus 1/T (Kelvin).Dissociation Rate Constants—To measure the dissociation rate constants, a complex of eIFiso4E or eIFiso4F with VPg was rapidly diluted 15-fold in a spectrofluorimeter cuvette, and the resulting increase in fluorescence was measured. Because of the high binding affinity of the protein-protein complex, a large dilution, which could not be accomplished by stopped-flow, was necessary. The concentration of the reactants before mixing was 10 μm VPg and 2 μm (each) eIFiso4E and eIFiso4F, respectively. The dissociation rates were determined from fits of the appropriate equations to the data using nonlinear least-square fitting program KaleidaGraph software (Version 2.1.3, Abelbeck software).RESULTSFluorescence Titration of eIFiso4E and eIFiso4F with VPg—To determine the binding constants for the viral protein linked to the genome (VPg) of TuMV and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F, direct fluorescence titration studies were performed as shown in Fig. 1. After the inner-filter effect corrections, we observed a total percent quench 58 and 50% for eIFiso4E and eIFiso4F fluorescence upon the addition of VPg at the highest molar ratios. The inset in Fig. 1 shows the corresponding Scatchard plots. The slope and intercept of the straight line obtained on the plot Q/[VPg] × 10–6 versus Q provided the binding constant (Ka) and binding capacity (n) of translation initiation factors for viral genome-linked protein (VPg). Kd values were obtained using a non-linear least squares analysis described elsewhere (35Khan M.A. Goss D.J. Biochemistry. 2004; 43: 9092-9097Crossref PubMed Scopus (23) Google Scholar). eIFiso4F exhibited almost 1.5 times stronger binding affinity than eIFiso4E (eIFiso4F, Kd = 81.31 ± 1.9 nm; eIFiso4E, Kd = 117.48 ± 2.7 nm). Similarly, the average binding sites (n) of eIFiso4E and eIFiso4F for VPg were determined to be 0.98 ± 0.15. Scatchard analysis of the binding data (Fig. 1) suggests that eIFiso4E and eIFiso4F each bound a single VPg.Fig. 2 shows a representative plot of eIFiso4E and eIFiso4F with VPg for determination of the dissociation constant (Kd). The titration curve in Fig. 2 shows the difference in fluorescence intensity between the protein-protein complex and the sum of the individual fluorescence intensities. From such analysis, the equilibrium binding constant can be calculated for the interactions between eIFiso4E or eIFiso4F with VPg (Table 1). eIFiso4E showed a strong interaction with VPg in the presence of eIFiso4G. No interaction was observed between eIFiso4G and VPg. A comparison of the dissociation constants at different temperatures is shown in Table 1. eIFiso4F has a higher affinity for VPg with a dissociation constant of 62.43 ± 0.61 nm as compared with eIFiso4E (97.47 ± 1.6 nm) at 22 °C. From the temperature dependence of these dissociation constants, the thermodynamic parameters for eIFiso4E and eIFiso4F with VPg were calculated (Table 2). A von't Hoff plot of –ln Keq versus the reciprocal of temperature (T –1) was used to calculate the thermodynamic parameters, entropy (ΔS°), and enthalpy (ΔH°). Fig. 3 shows the van't Hoff plot based on VPg binding to eIFiso4E or eIFiso4F; the values of ΔH° and ΔS° were obtained from the intercept and slope, respectively (correlation coefficient of >0.95). The van't Hoff analyses showed that the VPg binding is enthalpy-driven and entropy-favorable with a large negative ΔH°(–29.32 ± 0.13 kJmol–1) and positive ΔS° (36.88 ± 0.25 Jmol–1K–1) for eIFiso4E. The ΔG° values at 25 °C were calculated from equation, ΔG° = –RT ln Keq. The ΔG° values for eIFiso4E (–39.53 ± 0.32 kJmol–1) and eIFiso4F (–40.45 ± 0.51 kJmol–1) with VPg were shown in Table 2. Although the ΔG° value for the binding of VPg to eIFiso4E and eIFiso4F was almost the same, the data suggest different forces driving the interaction. The binding of eIFiso4E-VPg and eIFiso4F-VPg occurs as enthalpy-driven with large negative ΔH° and large positive ΔS°, leading to a negative ΔG° (Table 2). However, the activation energy for eIFiso4F is substantially larger, suggesting an energetically less favorable transition state.FIGURE 2Representative fluorescence intensity measurements on binding of eIFiso4E (○) and eIFiso4F (▵) to VPg. eIFiso4E and eIFiso4F, each at an initial concentration of 500 nm, were titrated by VPg. The curves shown are for the same concentrations to give an accurate comparison. The excitation wavelength was 280 nm, and the emission was 332 nm. The fluorescence is normalized by using the formula fb = (F0 – Fobs)/(Fobs – F∞)(F0/F∞) + (F0 – Fobs), which is directly related to the fraction of protein bound (fb). The solid lines are the fitted curves.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Equilibrium dissociation constants for the interaction of eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) with VPg, determined by fluorescence titrationComplexKd at 5 °CKd at 10 °CKd at 15 °CKd at 22 °CKd at 25 °CKd at 32 °CnmnmnmnmnmnmeIFiso4E-VPg47.09 ± 0.6558.16 ± 0.5170.89 ± 0.7397.47 ± 1.6117.48 ± 2.7137.59 ± 0.93eIFiso4F-VPg30.52 ± 0.3639.19 ± 0.2947.87 ± 0.4462.43 ± 0.681.31 ± 1.997.76 ± 0.58eIFiso4E-Ant-m7GTPaValues were obtained from Khan and Goss (35). ND, not determined. Dissociation constants were obtained by titrating eIFiso4E or eIFiso4F (500 nm) with VPg (0-1000 nm). The excitation wavelength was 280 nm, and fluorescence emission was 332 nm
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