14-3-3 Protein C-terminal Stretch Occupies Ligand Binding Groove and Is Displaced by Phosphopeptide Binding
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m408671200
ISSN1083-351X
AutoresJan Šilhán, Veronika Obšilová, Jaroslav Večeř, Petr Heřman, Miroslav Šulc, Jan Teisinger, Tomáš Obšil,
Tópico(s)Microbial Natural Products and Biosynthesis
Resumo14-3-3 proteins are important regulators of numerous cellular signaling circuits. They bind to phosphorylated protein ligands and regulate their functions by a number of different mechanisms. The C-terminal part of the 14-3-3 protein is known to be involved in the regulation of 14-3-3 binding properties. The structure of this region is unknown; however, a possible location of the C-terminal stretch within the ligand binding groove of the 14-3-3 protein has been suggested. To fully understand the role of the C-terminal stretch in the regulation of the 14-3-3 protein binding properties, we investigated the physical location of the C-terminal stretch and its changes upon the ligand binding. For this purpose, we have used Förster resonance energy transfer (FRET) measurements and molecular dynamics simulation. FRET measurements between Trp242 located at the end of the C-terminal stretch and a dansyl group attached at two different cysteine residues (Cys25 or Cys189) indicated that in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of 14-3-3 protein. Our data also showed that phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove. Intramolecular distances calculated from FRET measurements fit well with distances obtained from molecular dynamics simulation of full-length 14-3-3ζ protein. 14-3-3 proteins are important regulators of numerous cellular signaling circuits. They bind to phosphorylated protein ligands and regulate their functions by a number of different mechanisms. The C-terminal part of the 14-3-3 protein is known to be involved in the regulation of 14-3-3 binding properties. The structure of this region is unknown; however, a possible location of the C-terminal stretch within the ligand binding groove of the 14-3-3 protein has been suggested. To fully understand the role of the C-terminal stretch in the regulation of the 14-3-3 protein binding properties, we investigated the physical location of the C-terminal stretch and its changes upon the ligand binding. For this purpose, we have used Förster resonance energy transfer (FRET) measurements and molecular dynamics simulation. FRET measurements between Trp242 located at the end of the C-terminal stretch and a dansyl group attached at two different cysteine residues (Cys25 or Cys189) indicated that in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of 14-3-3 protein. Our data also showed that phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove. Intramolecular distances calculated from FRET measurements fit well with distances obtained from molecular dynamics simulation of full-length 14-3-3ζ protein. The 14-3-3 protein family represents one of the most important group among proteins recognizing phosphorylated targets (1Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1328) Google Scholar, 2Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1188) Google Scholar, 3Yaffe M.B. Elia A.E. Curr. Opin. Cell Biol. 2001; 13: 131-138Crossref PubMed Scopus (289) Google Scholar, 4Bridges D. Moorhead G.B. Science's STKE. 2004; (http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;stke.2422004re10)PubMed Google Scholar). Two canonical 14-3-3 binding motifs have been defined, RSXpSXP and RX(Y/F)XpSXP (5Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1348) Google Scholar, 6Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar), where pS denotes phosphoserine. Many of the 14-3-3 protein binding partners identified so far contain one of these motifs. Through the functional modulation of a wide range of binding partners, 14-3-3 proteins are involved in many biologically important processes, including cell cycle regulation, metabolism control, apoptosis, and control of gene transcription (1Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1328) Google Scholar, 2Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1188) Google Scholar, 3Yaffe M.B. Elia A.E. Curr. Opin. Cell Biol. 2001; 13: 131-138Crossref PubMed Scopus (289) Google Scholar, 4Bridges D. Moorhead G.B. Science's STKE. 2004; (http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;stke.2422004re10)PubMed Google Scholar). All 14-3-3 proteins form very stable homo- and heterodimers with characteristic cup-like shape and a large, 40-Å-wide, deep central channel (5Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1348) Google Scholar, 6Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 7Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (440) Google Scholar, 8Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. 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Uchiyama J. Kunihiro O. Ito M. Horigome T. Omata S. Shinkai F. Kaji H. Isobe T. J. Biol. Chem. 1995; 270: 28515-28518Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 13Liu Y.C. Elly C. Yoshida H. Bonnefoy-Berard N. Altman A. J. Biol. Chem. 1996; 271: 14591-14595Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Truong A.B. Masters S.C. Yang H. Fu H. Proteins. 2002; 49: 321-325Crossref PubMed Scopus (47) Google Scholar, 15Shen W. Clark A.C. Huber S.C. Plant J. 2003; 34: 473-484Crossref PubMed Scopus (32) Google Scholar, 16Obsilova V. Herman P. Vecer J. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 4531-4540Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In addition, the C-terminal stretch of two vertebrate 14-3-3 isoforms (τ and ζ) contains a casein kinase Iα phosphorylation site at position 232 (17Dubois T. Rommel C. Howell S. Steinhussen U. Soneji Y. Morrice N. Moelling K. Aitken A. J. Biol. Chem. 1997; 272: 28882-28888Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Phosphorylation of 14-3-3 proteins has been suggested to be an important regulatory mechanism of individual isoforms, and it has been shown that in vivo phosphorylation of the C-terminal phosphorylation site inhibits the interaction between 14-3-3ζ and Raf-1 kinase (17Dubois T. Rommel C. Howell S. Steinhussen U. Soneji Y. Morrice N. Moelling K. Aitken A. J. Biol. Chem. 1997; 272: 28882-28888Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 18Rommel C. Radziwill G. Lovric J. Noeldeke J. Heinicke T. Jones D. Aitken A. Moelling K. Oncogene. 1996; 12: 609-619PubMed Google Scholar). The structure of the C terminus is unknown, because this region is disordered in all available 14-3-3 protein crystal structures (5Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1348) Google Scholar, 6Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 7Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (440) Google Scholar, 8Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (400) Google Scholar, 9Obsil T. Ghirlando R. Klein D.C. Ganguly S. Dyda F. Cell. 2001; 105: 257-267Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 10Wurtele M. Jelich-Ottmann C. Wittinghofer A. Oecking C. EMBO J. 2003; 22: 987-994Crossref PubMed Scopus (272) Google Scholar). Liu et al. (7Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (440) Google Scholar) suggest that, in the absence of the ligand, the 14-3-3 protein C-terminal stretch could occupy the ligand binding groove and thus has to be pushed away during the ligand binding. Moreover, Truong et al. (14Truong A.B. Masters S.C. Yang H. Fu H. Proteins. 2002; 49: 321-325Crossref PubMed Scopus (47) Google Scholar) show that removal of the C terminus increases the binding affinity of 14-3-3ζ protein for several tested ligands and propose that the C terminus might function as an autoinhibitor by suppressing unspecific interactions between 14-3-3 protein and inappropriate ligands. In addition, we have recently shown that phosphopeptide binding changes the conformation and increases the flexibility of the 14-3-3 protein C-terminal stretch (16Obsilova V. Herman P. Vecer J. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 4531-4540Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To fully understand the role of the C-terminal stretch in the regulation of 14-3-3 protein binding properties, we have attempted to provide evidence for the location of the missing C-terminal part of the 14-3-3 protein molecule. Förster resonance energy transfer (FRET) 1The abbreviations used are: FRET, Förster resonance energy transfer; 1,5-IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid; AEDANS, 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid; pRaf-259, phosphorylated peptide Raf-259; MD, molecular dynamics; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. measurements between Trp242 (located within the C-terminal stretch) and a dansyl group (attached at two different cysteine residues) indicate that, in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of the 14-3-3 protein. Our data also showed that phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove. Intramolecular distances calculated from FRET measurements fit well with distances obtained from molecular dynamics simulation of full-length 14-3-3ζ protein. Expression and Purification of 14-3-3 Proteins—All 14-3-3 protein mutants were generated using the QuikChange kit (Stratagene). The 14-3-3 proteins were expressed and purified as described by Obsilova et al. (16Obsilova V. Herman P. Vecer J. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 4531-4540Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Labeling of 14-3-3 Protein Mutants by 1,5-IAEDANS—Human 14-3-3ζ protein possesses three cysteine residues (Cys25, Cys94, and Cys189). To prepare proteins suitable for FRET measurements, we constructed three mutants containing a single cysteine residue (either at position 25, 94, or 189, respectively) and a single Trp residue located at the end of the C-terminal stretch at position 242. Covalent modification of the 14-3-3ζ protein containing the single Trp242 and single Cys residues either at position 25, 94, or 189, respectively, with 1,5-IAEDANS was carried out as described previously (19Polit A. Blaszczyk U. Wasylewski Z. Eur. J. Biochem. 2003; 270: 1413-1423Crossref PubMed Scopus (15) Google Scholar). Briefly, the protein (50–70 μm) in 50 mm Tris (pH 7.5), 100 mm NaCl, and 1 mm EDTA and label were mixed at a molar ratio of 1:40 and incubated at 30 °C for 2 h and then at 4 °C overnight in the dark. The free unreacted label was removed by gel filtration in buffer containing 50 mm Tris (pH 7.5), 100 mm NaCl, and 1 mm EDTA. The incorporation stoichiometry was determined by the absorbance at 336 nm using an extinction coefficient of 5700 m-1 cm-1 (Molecular Probes, Eugene, OR). Mass Spectrometry Analysis—MALDI-TOF mass spectrometry was used to check amino acid sequences of 14-3-3 protein constructs with mutated cysteines and verify the modification of cysteine residues by the AEDANS moiety. Samples were first separated by 12% SDS-PAGE, and the excised protein bands were digested directly in gel (20Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7822) Google Scholar). The resulting peptide mixtures were extracted by 30% acetonitrile and 0.5% acetic acid and subjected to the MALDI-TOF mass spectrometer BI-FLEX (Bruker-Franzen, Bremen, Germany) equipped with a nitrogen laser (337 nm) and gridless delayed extraction ion source. Ion acceleration voltage was 19 kV, and the reflectron voltage was set to 20 kV. Spectra were calibrated externally using the monoisotopic [M+H]+ ion of peptide standards angiotensin I (Sigma). A saturated solution of α-cyano-4-hydroxy-cinnamic acid in 30% MeCN 0.3% acetic acid was used as a MALDI matrix. A 1-μl sample was loaded on the target, and the droplet was allowed to dry at ambient temperature, overlaid with 1 μl of matrix solution, and then allowed to co-crystallize at ambient temperature. Negative and positive ion mass spectra of peptide maps were measured in the reflection mode to check amino acid sequences of tryptic peptides with mutated cysteines (Cys25 or Cys189) in used 14-3-3ζ protein mutants and to control the labeling of proteins with 1,5-IAEDANS. The detected peak in negative ion mass spectra having the mass of 896.4 (m/z) corresponds to peptide ACSLAK (590.3 m/z) with labeled Cys189 by 1,5-IAEDANS, and similarly, the peak of 1351.6 (m/z) fits to peptide YDDMAACMK (1045.4 m/z) with labeled Cys25 by 1,5-IAEDANS. The identity and structure of these labeled tryptic peptides were further corroborated by analysis of their post-source decay spectra (data not shown). Steady-state Fluorescence Measurements—Fluorescence spectra were taken on a PerkinElmer LS50B spectrofluorometer at 22 °C with 5 μm protein in 50 mm Tris (pH 7.5), 100 mm NaCl, and 1 mm EDTA in 10-mm cells. Excitation was done at 295 nm; the bandwidths were 5 nm for both excitation and emission. Synthetic peptides LSQRQRSTSTPNVHMV (Raf-259) or LSQRQRSTpSTPNVHMV (pRaf-259), where pS denotes phosphoserine (PolyPeptide Laboratories, Prague, Czech Republic) dissolved in 50 mm Tris-HCl (pH 7.5), 100 mm NaCl buffer were used as 14-3-3 ligands. 14-3-3 protein concentration was 6 μm, and ligand (pRaf-259 or Raf-259 peptide) concentration was 25 μm. Fluorescence Peptide Binding Assay—Fluorescence energy transfer between AEDANS and fluorescein has been used to determine the dissociation constant of peptide binding to AEDANS-labeled 14-3-3ζ protein mutants (21Zhang H. Liu X.H. Zhang K. Chen C.K. Frederick J.M. Prestwich G.D. Baehr W. J. Biol. Chem. 2004; 279: 407-413Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Fluorescence measurements were performed using a PerkinElmer LS50B spectrofluorometer. Peptides Raf-259 and pRaf-259 with a fluorescein group attached to the N terminus (PolyPeptide Laboratories, Prague, Czech Republic) were titrated into the cuvette containing 400 nm 14-3-3ζ protein mutants labeled by 1,5-IAEDANS in 50 mm Tris (pH 7.5), 100 mm NaCl, and 1 mm EDTA buffer. After mixing, the samples were incubated for 1 min at room temperature, and then the dansyl fluorescence at 480 nm was recorded using an excitation wavelength of 336 nm. The relative change in the fluorescence signal, fB, was plotted as a function of peptide concentration and was fitted to Equation 1 (22Kohler J.J. Schepartz A. Biochemistry. 2001; 40: 130-142Crossref PubMed Scopus (99) Google Scholar) to determine the KD values, fB={1/(2[14-3-3])}{KD+[14-3-3]+[Pep]−(KD+[14-3-3]+[Pep])2−4[14-3-3][Pep]} Eq.1 where KD is the equilibrium dissociation constant. Nonlinear data fitting was performed using the Origin 6.0 software package (Microcal Software Inc.). Time-resolved Fluorescence Measurements—Fluorescence resonance energy transfer was observed between the single tryptophan residue Trp242 and the AEDANS moiety covalently attached to Cys25 or Cys189. Fluorescence intensity decays of Trp242 were measured on an apparatus as described by Obsilova et al. (16Obsilova V. Herman P. Vecer J. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 4531-4540Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Fluorescence decays were acquired under"magic angle"conditions, where the measured intensity decay I(t) was independent of a rotational diffusion of the chromophore and provided unbiased information about lifetimes. The apparatus response function was done at the excitation wavelength measured with a diluted Ludox solution. The samples were placed in a thermostatic holder, and all experiments were performed at 22 °C in buffer containing 50 mm Tris-HCl (pH 7.5), 100 mm NaCl, and 1 mm EDTA. 14-3-3 protein concentration was 20 μm, and ligand (pRaf-259 or Raf-259 peptide) concentration was 50 μm. Fluorescence data processing was performed as described previously using the singular value decomposition maximum entropy method (16Obsilova V. Herman P. Vecer J. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 4531-4540Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 23Bryan R.K. Eur. Biophys. J. 1990; 18: 165-174Crossref Scopus (259) Google Scholar, 24Brochon J.C. Fluorescence Anisotropy Decay Analysis by Quantified Maximum Entropy Method, User Manual for PULSE5QT Program. MaxEnt Solutions, Ltd., Cambridge, UK1999: 33Google Scholar, 25Brochon J.C. Methods Enzymol. 1994; 240: 262-311Crossref PubMed Scopus (254) Google Scholar), and the mean lifetimes were calculated. The average efficiency of energy transfer E was calculated from the mean donor lifetime in the presence (τDA) and absence of acceptor (τD). E=1−(τDA/τD) The average distance between the donor-acceptor pair R was calculated from Equation 3, R=R0·61/E−1 Eq.3 where R0 is the Förster critical distance (the distance at which the energy transfer occurs with 50% efficiency). R0 is given by (26Wu P. Brand L. Anal. Biochem. 1994; 218: 1-13Crossref PubMed Scopus (1148) Google Scholar, 27Lakowicz J.R. Principles of Fluorescence Spectroscopy. Kluwer Academic, Plenum Publishing Corp., New York1999Crossref Google Scholar), R0=[8.8×1023κ2n−4QDJ(λ)]1/6[Å] Eq.4 where n is the refractive index of the medium, QD is the emission quantum yield of the donor in the absence of the acceptor, and κ2 is the orientation factor that accounts for relative orientation of the donor emission and acceptor absorption transition dipole. The spectral overlap integral J(λ) of the donor fluorescence and acceptor absorption was calculated from Equation 5 (26Wu P. Brand L. Anal. Biochem. 1994; 218: 1-13Crossref PubMed Scopus (1148) Google Scholar, 27Lakowicz J.R. Principles of Fluorescence Spectroscopy. Kluwer Academic, Plenum Publishing Corp., New York1999Crossref Google Scholar), J(λ)=∫FD(λ)εa(λ)λ4dλ/∫FD(λ)dλ{\\ }[cm3M−1] Eq.5 where FD(λ) and ϵa(λ) represent the fluorescence intensity of the donor and the molar extinction coefficient of the acceptor, respectively, at the wavelength λ expressed in centimeters. The orientation factor κ2 was assumed to be equal to ⅔, which corresponds to randomly oriented dipole moments of donor-acceptor pairs. A value of 1.31 was used for the refractive index of the aqueous solution. Quantum yields of the donor, which is the Trp242 in the single Trp mutant of the 14-3-3ζ protein, were calculated relative to the QD of L-Trp in water (28Chen R.F. Anal. Lett. 1967; 1: 35-42Crossref Scopus (413) Google Scholar). Molecular Modeling—Molecular dynamics (MD) simulations were performed according to the protocol published previously (16Obsilova V. Herman P. Vecer J. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 4531-4540Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The GROMACS version 3.1 (www.gromacs.org) molecular dynamics software package was used with the parameter set ffG43a1p (29Hansson T. Nordlund P. Aqvist J. J. Mol. Biol. 1997; 265: 118-127Crossref PubMed Scopus (73) Google Scholar). All MD trajectories were analyzed using GROMACS software. The cluster analysis was performed using the GROMOS software method (30Daura X. Gademann K. Jaun B. Seebach D. Van Gunsteren W.F. Mark A.E. Angew. Chem. Int. Ed. Engl. 1999; 38: 236-240Crossref Google Scholar). A model of the 14-3-3ζ protein with attached AEDANS moieties at residues Cys25 and Cys189 was built using a representative conformation of 14-3-3ζ protein (obtained using a cluster analysis of simulated trajectory with a root mean square deviation cutoff of 1 Å) and AEDANS coordinates obtained from the crystal structure of labeled RNase (Protein Data Bank accession code 1RAS) (31Baudet-Nessler S. Jullien M. Crosio M.P. Janin J. Biochemistry. 1993; 32: 8457-8464Crossref PubMed Scopus (17) Google Scholar). The geometry of the resulting complex was optimized by energy minimization using the molecular modeling package Ghemical (32Hassinen T. Peräkylä M. J. Comput. Chem. 2001; 22: 1229-1242Crossref Scopus (119) Google Scholar) and Desktop Molecular Modeler version 4.2 (Polyhedron Software). Construction of 14-3-3ζ Protein Mutants for Förster Resonance Energy Transfer—To provide evidence for the physical location of the C-terminal stretch using FRET, we have constructed three 14-3-3ζ protein mutants containing a single Trp residue within the C-terminal stretch at the position 242 (residues Trp59 and Trp228 were mutated to Phe) and a single Cys residue located at three different positions: Cys25 (this mutant was named 14-3-3w242c25), Cys94 (14-3-3w242c94), and Cys189 (14-3-3w242c189). The Trp residue served as an energy transfer donor, and the Cys residues were selectively labeled with the extrinsic fluorophore 1,5-IAEDANS as energy acceptors. Labeling experiments revealed that only Cys25 and Cys189 can be completely modified by 1,5-IAEDANS. The stoichiometries of AEDANS/mol of these two 14-3-3ζ protein mutants were found to be ∼1 (0.95 and 1.06, respectively). The third cysteine, Cys94, was modified partially by 1,5-IAEDANS (labeling stoichiometry was found to be only ∼0.4) and therefore was not used for FRET measurements. Modification of 14-3-3ζ protein mutants by 1,5-IAEDANS has been verified using MALDI-TOF mass spectrometry. A fluorescence binding assay was used to test the binding abilities of both 14-3-3ζ protein mutants (Fig. 1). These experiments revealed that both 14-3-3ζ protein mutants modified with 1,5-IAEDANS bind phosphorylated pRaf-259 peptide with comparable affinity as wild-type protein, which binds pRaf-259 peptide with KD value of 116 nm (5Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1348) Google Scholar). No significant binding of unphosphorylated Raf-259 peptide within the used concentration range has been detected (data not shown). Investigation of the C-terminal Stretch Location Using FRET Steady-state Fluorescence Measurements—FRET measurements between Trp242 and AEDANS moiety attached to a cysteine residue of 14-3-3ζ protein mutants were used to investigate the physical location of the C-terminal stretch of the 14-3-3ζ protein. First, we tested the presence of the energy transfer between Trp242 and AEDANS moieties using the steady-state fluorescence. Tryptophan fluorescence emission spectra of 14-3-3ζ protein mutants, unmodified and modified with 1,5-IAEDANS, are shown in Fig. 2. When excited at 295 nm, Trp242 has a fluorescence emission maximum near 350 nm. In the presence of 1,5-IAEDANS, Trp242 fluorescence intensities of both single cysteine-containing mutants were significantly reduced compared with unmodified proteins, indicating the presence of FRET. The sensitized fluorescence emission of AEDANS (induced by the nonradiative transfer of energy from Trp242) was observed as a peak at 485 nm. In the case of the 14-3-3w242c25 mutant, the donor steady-state fluorescence was quenched by ∼24% compared with the emission of unlabeled protein (Fig. 2A). Higher steady-state FRET efficiency was observed for the 14-3-3w242c189 mutant, with ∼50% reduction of the donor fluorescence (Fig. 2C), indicating that AEDANS attached to Cys189 is closer to Trp242 than in the case of Cys25. Next, we tested the effect of the peptide binding on steady-state FRET efficiency between Trp242 and AEDANS. The presence of the unphosphorylated peptide Raf-259 had no effect on steady-state FRET efficiency, irrespective of AEDANS position within the 14-3-3 protein molecule (data not shown). On the other hand, the addition of the phosphorylated peptide pRaf-259, which contains optimal 14-3-3 protein binding motif and binds to 14-3-3ζ protein with high affinity, was found to affect steady-state FRET efficiency. The effect was dependent on the position of AEDANS. In the case of the 14-3-3w242c25 mutant, the pRaf-259 binding slightly reduced FRET efficiency (compare Fig. 2, A and B), whereas for the 14-3-3w242c189 mutant, the FRET remained practically unchanged (compare Fig. 2, C and D). Investigation of the C-terminal Stretch Location Using FRET Time-resolved Fluorescence Measurements—To quantitatively investigate the physical location of the C-terminal stretch, we employed time-resolved intensity decays of Trp242 to measure distances between residue Trp242 and an AEDANS moiety attached at different positions within the 14-3-3ζ protein molecule. We chose the time-resolved fluorescence approach, because, unlike fluorescence intensities, lifetimes do not depend on the excitation intensity, excited sample volume, protein concentration, or photobleaching, etc. Consequently, time-resolved data are more reliable than and superior to fluorescence intensities for quantitative FRET measurements. To use FRET to measure distances between Trp242 and AEDANS, the Förster critical distance R0 of this donor-acceptor pair must be determined first. The R0 is a distance between the donor and acceptor at which the energy transfer is 50% efficient. The R0 was determined from Equations 4 and 5. The spectral overlap J(λ) between Trp242 and AEDANS (obtained by numerical integration of a product of an area-normalized emission spectrum of 14-3-3w242 protein containing a single tryptophan at position 242 and the absorption spectrum of AEDANS) was found to be 5.876 × 10-15 cm3m-1. The quantum yield of Trp242 fluorescence of 14-3-3ζ protein mutants at 22 °C was determined to be 0.074 ± 0.002 in the absence of the ligand, 0.071 ± 0.002 in the presence of 25 μm pRaf-259 peptide, and 0.073 ± 0.003 in the presence of 25 μm Raf-259 peptide, respectively. The quantum yields were determined relative to a standard solution of tryptophan in water (QD = 0.140) (28Chen R.F. Anal. Lett. 1967; 1: 35-42Crossref Scopus (413) Google Scholar). This resulted in the calculated R0 of 19.7 Å in both the absence of the ligand and the presence of unphosphorylated peptide Raf-259. In the presence of the phosphorylated peptide pRaf-259, the R0 is 19.5 Å. These R0 distances are somewhat lower compared with the value of 22 Å reported elsewhere (19Polit A. Blaszczyk U. Wasylewski Z. Eur. J. Biochem. 2003; 270: 1413-1423Crossref PubMed Scopus (15) Google Scholar), presumably because of the lower quantum yield of Trp242 fluorescence. Time-resolved fluorescence intensity decays were analyzed using a singular value decomposition maximum entropy method as described previously (16Obsilova V. Herman P. Vecer J. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 4531-4540Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The intensity decays of Trp242 for all three 14-3-3ζ protein mutants can be adequately described by a lifetime distribution containing three lifetime components (data not shown). Mean excited-state lifetimes (τmean) of Trp242 in the absence and presence of acceptor and the efficiency of the energy transfer are presented in Table I. Upon labeling by 1,5-IAEDANS for both mutants, the τmean of donor was reduced as a result of the energy transfer (Fig. 3). Values of the FRET efficiency were used to calculate the average distances between the donor and the acceptors. Distances between Trp242 and AEDANS attached at Cys25 and Cys189 were calculated to be 30.4 Å and 23.8 Å, respectively. These distances strongly indicate that, in the absence of the ligand, the C-terminal stretch of 14-3-3ζ protein is physically located within the ligand binding groove (see "Molecular Modeling" for details).Table ISummary of energy transfer measurements14-3-3ζ MutantLabelLigandτmeanaMean lifetimes were calculated as(τmean=∑ifiτi), where fi is an intensity fraction of the ith lifetime component τi,bStandard deviation is 0.01 nsEcE, energy transferR0dF
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