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• W2000435338 abstract "In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly67 amide nitrogen to the Ser65 carbonyl carbon is catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg96 and Glu222 in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant = 7.5 × 103 h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9–10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h (pH 7–10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu222 carboxylate titrates with an apparent pKa of 6.5 in R96M and that the Gly67 amide nitrogen titrates with an apparent pKaa of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu222 plays the role of a general base, facilitating proton abstraction from the Gly67 amide nitrogen or the Tyr66 α-carbon. Arg96 fulfills the role of an electrophile by lowering the respective pK values and stabilizing the α-enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events. In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly67 amide nitrogen to the Ser65 carbonyl carbon is catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg96 and Glu222 in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant = 7.5 × 103 h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9–10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h (pH 7–10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu222 carboxylate titrates with an apparent pKa of 6.5 in R96M and that the Gly67 amide nitrogen titrates with an apparent pKaa of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu222 plays the role of a general base, facilitating proton abstraction from the Gly67 amide nitrogen or the Tyr66 α-carbon. Arg96 fulfills the role of an electrophile by lowering the respective pK values and stabilizing the α-enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events. Green fluorescent protein (GFP) 1The abbreviations used are: GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein (GFP-F64L/S65T); PIPES, 1,4-piperazinediethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; HPLC, high-pressure liquid chromatography; MALDI, matrix-assisted laser desorption ionization; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone. 1The abbreviations used are: GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein (GFP-F64L/S65T); PIPES, 1,4-piperazinediethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; HPLC, high-pressure liquid chromatography; MALDI, matrix-assisted laser desorption ionization; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone. and its homologues constitute a family of small compact 11-stranded β-barrel proteins that generate their own fluorophore in an autocatalytic fashion (1Ormo M. Cubitt A.B. Kallio K. Gross L.A. Tsien R.Y. Remington S.J. Science. 1996; 273: 1392-1395Crossref PubMed Scopus (1902) Google Scholar, 2Yang F. Moss L.G. Phillips G.N. Nature Biotechnol. 1996; 14: 1246-1251Crossref PubMed Scopus (1286) Google Scholar, 3Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar). Because of their bright colors ranging from cyan to green to red (4Matz M.V. Fradkov A.F. Labas Y.A. Savitsky A.P. Zaraisky A.G. Markelov M.L. Lukyanov S.A. Nature Biotechnol. 1999; 17: 969-973Crossref PubMed Scopus (1501) Google Scholar, 5Lukyanov K.A. Fradkov A.F. Gurskaya N.G. Matz M.V. Labas Y.A. Savitsky A.P. Markelov M.L. Zaraisky A.G. Zhao X. Fang Y. Tan W. Lukyanov S.A. J. Biol. Chem. 2000; 275: 25879-25882Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 6Matz M.V. Lukyanov K.A. Lukyanov S.A. BioEssays. 2002; 24: 953-959Crossref PubMed Scopus (129) Google Scholar), these proteins are used extensively as research tools in molecular and cell biology (3Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar, 7Verkhusha V. Lukyanov K.A. Nat. Biotechnol. 2004; 22: 289-296Crossref PubMed Scopus (273) Google Scholar). The various colors originate from a fluorescent entity generated in the interior of the protein by the spontaneous covalent modification of three amino acid residues that are part of the polypeptide chain of the protein. Chromophore formation follows protein folding and is initiated by a main-chain condensation reaction, generating a five-membered heterocycle from the backbone atoms of the original polypeptide chain (Ser65, Tyr66, and Gly67 in wild-type GFP) (Scheme 1) (8Cubitt A.B. Heim R. Adams S.R. Boyd A.E. Gross L.A. Tsien R.Y. Trends Biochem. Sci. 1995; 20: 448-455Abstract Full Text PDF PubMed Scopus (1193) Google Scholar, 9Heim R. Prasher D.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12501-12504Crossref PubMed Scopus (1476) Google Scholar). Recent x-ray structures of GFP variants (10Barondeau D.P. Putnam C.D. Kassmann C.J. Tainer J.A. Getzoff E.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12111-12116Crossref PubMed Scopus (159) Google Scholar, 11Barondeau D.P. Kassmann C.J. Tainer J.A. Getzoff E.D. Biochemistry. 2005; 44: 1960-1970Crossref PubMed Scopus (66) Google Scholar) have confirmed that peptide cyclization is the first chemical step in chromophore formation, as originally proposed (8Cubitt A.B. Heim R. Adams S.R. Boyd A.E. Gross L.A. Tsien R.Y. Trends Biochem. Sci. 1995; 20: 448-455Abstract Full Text PDF PubMed Scopus (1193) Google Scholar), and is responsible for triggering a series of downstream processing events that include air oxidation. Among all of the GFP-like proteins (7Verkhusha V. Lukyanov K.A. Nat. Biotechnol. 2004; 22: 289-296Crossref PubMed Scopus (273) Google Scholar), GFP itself comprises the simplest system with the least number of chemical steps leading to the mature chromophore, and the mechanism has generally been written as a cyclization-dehydration-oxidation reaction (Scheme 1, Mechanism A) (8Cubitt A.B. Heim R. Adams S.R. Boyd A.E. Gross L.A. Tsien R.Y. Trends Biochem. Sci. 1995; 20: 448-455Abstract Full Text PDF PubMed Scopus (1193) Google Scholar). Based on the x-ray structure of Y66L (12Rosenow M.A. Huffman H.A. Phail M.E. Wachter R.M. Biochemistry. 2004; 43: 4464-4472Crossref PubMed Scopus (80) Google Scholar), we have recently proposed a modified mechanism consisting of the sequence cyclization-oxidation-dehydration (Scheme 1, Mechanism B), in accord with oxidative stabilization of the cyclic tetrahedral intermediate. It is possible that both pathways A and B occur in GFP maturation and that the relative rates of the individual steps are altered by the mutant forms. In many GFP variants, the optical properties of the mature chromophore are pH-dependent. Absorbance around 400 nm excites the neutral chromophore, and absorbance around 480 nm excites the chromophore anion in which the phenolic end is deprotonated (Scheme 1) (3Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar). At physiological pH, enhanced green fluorescent protein EGFP (GFP-F64L/S65T) (13Cormack B.P. Valdivia R.H. Falkow S. Gene (Amst.). 1996; 173: 33-38Crossref PubMed Scopus (2489) Google Scholar) is brighter than wild-type GFP due to a predominantly anionic chromophore, similar to GFP-S65T in which the chromophore titrates with a pKa of 6.0 (14Elsliger M.-A. Wachter R.M. Hanson G.T. Kallio K. Remington S.J. Biochemistry. 1999; 38: 5296-5301Crossref PubMed Scopus (288) Google Scholar).The kinetics of de novo chromophore formation has been determined by monitoring the rate of fluorescence acquisition. The GFP-S65T maturation process, inclusive of protein folding and the ensuing chemical steps, has been shown to proceed in vitro with a time constant of 122 min (15Reid B.G. Flynn G.C. Biochemistry. 1997; 36: 6786-6791Crossref PubMed Scopus (356) Google Scholar). In this experiment, maturation was induced by rapid dilution of urea-solubilized inclusion bodies at room temperature. De novo protein folding was monitored independently by a trypsin resistance assay and was estimated to proceed with a time constant of 14 min (t½ ∼10 min).In a different type of experiment, Escherichia coli cultures expressing GFP were grown anaerobically and the protein was allowed to mature for 3 days in the absence of oxygen. Upon admission of air to the cells, the time course of subsequent acquisition of green fluorescence gave a time constant of 120 min for wild-type GFP and 27 min for GFP-S65T (9Heim R. Prasher D.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12501-12504Crossref PubMed Scopus (1476) Google Scholar, 16Heim R. Cubitt A.B. Tsien R.Y. Nature. 1995; 373: 663-664Crossref PubMed Scopus (1508) Google Scholar). These data are consistent with air oxidation as the rate-determining step in the overall maturation process (Scheme 1) (3Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar).Two residues in the immediate environment of the GFP chromophore are highly conserved, Arg96 and Glu222 (4Matz M.V. Fradkov A.F. Labas Y.A. Savitsky A.P. Zaraisky A.G. Markelov M.L. Lukyanov S.A. Nature Biotechnol. 1999; 17: 969-973Crossref PubMed Scopus (1501) Google Scholar). Proposed functions for these residues range from electrostatic, steric, and catalytic roles to contributions to protein folding and stability (3Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar, 17Branchini B.R. Nemser A.R. Zimmer M. J. Am. Chem. Soc. 1998; 120: 1-6Crossref Scopus (44) Google Scholar). Neither Arg96 nor Glu222 is absolutely necessary for chromophore biosynthesis (18Jung G. Wiehler J. Zumbusch A. Biophys. J. 2005; 88: 1932-1947Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). For example, GFP variants reported to exhibit altered spectral properties include the E222G mutant (19Ehrig T. O'Kane D.J. Prendergast F.G. FEBS Lett. 1995; 367: 163-166Crossref PubMed Scopus (139) Google Scholar) and the R96C mutant (3Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar). More recently, the substitution of Arg96 with an alanine was shown to result in extremely slow chromophore maturation on the order of 2 months at 30 °C (10Barondeau D.P. Putnam C.D. Kassmann C.J. Tainer J.A. Getzoff E.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12111-12116Crossref PubMed Scopus (159) Google Scholar). The hydrogen bond of the carbonyl oxygen of Tyr66 to the guanidinium group of Arg96 appears to aid in the cyclization reaction, and two similar mechanisms for catalysis by Arg96 have been put forth. These mechanisms emphasize polarization of the peptide bond between residues 66 and 67 (10Barondeau D.P. Putnam C.D. Kassmann C.J. Tainer J.A. Getzoff E.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12111-12116Crossref PubMed Scopus (159) Google Scholar) or suggest a proton transfer from the guanidinium group to the carbonyl oxygen of Tyr66 (20Siegbahn P.E.M. Wirstam M. Zimmer M. Int. J. Quant. Chem. 2001; 81: 169-186Crossref Scopus (29) Google Scholar). Both processes would increase the nucleophilic reactivity of the amide nitrogen of Gly67 to facilitate addition to the carbonyl carbon of residue 65 (Scheme 1).Here, we investigate the function of the buried and highly conserved residues, Arg96 and Glu222, in chromophore biosynthesis. As our parent clone, we chose EGFP, the enhanced green fluorescent protein originally selected based on its brightness (13Cormack B.P. Valdivia R.H. Falkow S. Gene (Amst.). 1996; 173: 33-38Crossref PubMed Scopus (2489) Google Scholar). Observed rate constants and extracted pKa values for chromophore formation in R96M and E222Q are in accord with a peptide cyclization reaction that partitions through an amide anion or an α-enolate species. Delineating the structural and catalytic requirements for efficient chromophore biosynthesis will be essential in the prediction of similar cross-linking chemistries in unrelated proteins.EXPERIMENTAL PROCEDURESSite-directed Mutagenesis, Protein Preparation, and Optical Characterization—Amino acid substitutions were introduced into EGFP using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene). The proteins, N-terminally tagged with a His6 tag in the plasmid pRSETB (Invitrogen), were overexpressed in E. coli strain JM109-DE3 and purified as described previously (1Ormo M. Cubitt A.B. Kallio K. Gross L.A. Tsien R.Y. Remington S.J. Science. 1996; 273: 1392-1395Crossref PubMed Scopus (1902) Google Scholar). Protein homogeneity was estimated to be 90% by SDS-PAGE. UV-visible absorbance spectra were collected on a Shimadzu UV-2401 spectrophotometer.Chromophore Formation Kinetics of R96M and E222Q—The R96M variant was purified following the standard 2-day procedure, because chromophore formation is not observed during this time. However, the E222Q variant was purified by a rapid 2-h procedure (21Remington S.J. Wachter R.M. Yarbrough D.K. Branchaud B.P. Anderson D.C. Kallio K. Lukyanov K.A. Biochemistry. 2005; 44: 202-212Crossref PubMed Scopus (121) Google Scholar) to prepare protein primarily in the precursor state at the onset of kinetic experiments. 250-ml E. coli cultures were induced for 2 h, the cells were disrupted with lysozyme, and the protein was purified via nickel-nitrilotriacetic acid at 4 °C. PD-10 Sephadex columns (Amersham Biosciences) were used to buffer-exchange the protein into 20 mm HEPES (pH 7.0), 300 mm NaCl, and 1 mm EDTA at 4 °C. Purified protein was immediately flash-frozen and stored at –80 °C. The extent of chromophore maturation was determined after base denaturation (see below).The rate of chromophore formation was monitored as a function of pH by determining the increase in visible absorbance with time. Experiments were initiated by 10-fold dilution of the protein into 20 mm buffer (PIPES, HEPES, CHES, or CAPS), 300 mm NaCl, and 1 mm EDTA. The maturation rate for each variant was monitored at pH 6.0, 7.0, 8.0, 9.0, 9.5, and 10.0 over a period of 10 h (E222Q, 0.1 mg/ml) or 2 months (R96M, 0.5 mg/ml) at 30 °C by incubation in a temperature-controlled chamber. The increase in absorbance of the chromophore anion (483 nm for E222Q) was plotted as a function of time and computer-fitted to a unimolecular reaction rate equation (Equation 1) using Kaleidagraph™, A=Amax−e(−kt)×Amax (Eq. 1) where A is the change in absorbance, Amax is the maximum absorbance for complete maturation, k = kobs = observed rate constant, and t is time. Direct monitoring of R96M chromophore formation via native-state absorbance at 384 nm (neutral state of the chromophore) was not feasible because of slow chromophore formation. Consequently, the percent chromophore was determined at each time point after base denaturation (see below). The extracted pseudo-first-order rate constants were plotted as a function of pH, and the data were computer-fitted to a rate expression for a base-catalyzed reaction with a rapid preliminary equilibrium to form the conjugate base of an ionizable group according to Equation 2, kobs=[k1×Ka]/[10(−pH)+Ka], (Eq. 2) where k1 is the pH-independent rate constant and Ka is the proton dissociation constant.Determination of Percent Chromophore via Base Denaturation—The concentration of purified protein was determined using the Advanced protein assay (Cytoskeleton, Inc., Denver, CO). An aliquot of the protein was then diluted 10-fold into 0.2 m NaOH, and the optical density at 448 nm (chromophore anion absorbance in the denatured state) was determined (22Ward W.W. DeLuca M.A. McElroy W.D. Bioluminescence and Chemiluminescence. Academic Press, New York1981: 235-242Crossref Google Scholar, 23Wachter R.M. Brett A.K. Heim R. Kallio K. Tsien R.Y. Boxer S.G. Remington S.J. Biochemistry. 1997; 36: 9759-9765Crossref PubMed Scopus (151) Google Scholar). For EGFP, the extinction coefficient in our preparations was calculated to be ϵ448 = 33,500 (±800, n = 5), somewhat below the literature values of ϵ448 = 44,000 for wild-type GFP (22Ward W.W. DeLuca M.A. McElroy W.D. Bioluminescence and Chemiluminescence. Academic Press, New York1981: 235-242Crossref Google Scholar) and ϵ425 = 41,000 for a synthetic model compound (24Niwa H. Inouye S. Hirano T. Matsuno T. Kojima S. Kubota M. Ohashi M. Tsuji F.I. Proc. Natl. Acad. Sci. (U. S. A.). 1996; 93: 13617-13622Crossref PubMed Scopus (395) Google Scholar). For each experiment, chromophore concentrations were determined in triplicate and compared with the protein concentration.Maturation Kinetics of EGFP Prepared from Inclusion Bodies—Inclusion bodies of EGFP were prepared and washed as described previously (12Rosenow M.A. Huffman H.A. Phail M.E. Wachter R.M. Biochemistry. 2004; 43: 4464-4472Crossref PubMed Scopus (80) Google Scholar) and then suspended in 50 mm Tris (pH 7.9), 500 mm NaCl, and 20 mm EDTA, quick-frozen in liquid nitrogen, and stored at –80 °C. Aliquots were thawed and centrifuged for 15 min at a relative centrifugal force of 20,800. The pellet was solubilized in 2 ml of urea buffer (50 mm HEPES (pH 7.9), 50 mm NaCl, 1 mm dithiothreitol, and 8 m urea), and the solution was clarified by centrifugation at a relative centrifugal force of 20,800 for 15 min, loaded onto a nickel-nitrilotriacetic acid affinity column (Qiagen), and washed with 50 mm HEPES (pH 7.9), 50 mm NaCl, 8 m urea, and 20 mm imidazole. Denatured EGFP was subsequently eluted by the addition of 100 mm imidazole to the wash buffer and incubated with 10 mm TCEP (tris-2-carboxyethyl phosphine) for 10 min to allow for the reduction of disulfide bonds. Protein folding was induced by rapid dilution. 45 μl of urea-solubilized EGFP was added to 860 μl of vigorously stirring folding buffer (333 mm NaCl, 1.1 mm EDTA, 1.1 mm TCEP, and 11 mm CHES (pH 9.0)) while maintaining the temperature at 30 °C by a circulating water bath. In some experiments, the pH was adjusted after 10 min by the addition of 200 μl of 500 mm buffer (PIPES (pH 6.0), HEPES (pH 7.0 or 8.0), CHES (pH 9.0), or CAPS (pH 10)). 100-μl aliquots were removed from the reaction at various time points and spin-filtered though a 0.1-μm centrifugal filter device (Millipore). Absorbance scans of the filtrate were collected from 600 to 240 nm, and the change in absorbance at 489 nm (native-state chromophore anion) was fitted to a unimolecular rate (Equation 1).Trypsinolysis and Peptide Purification—Proteolysis of EGFP and its variants was carried out according to the following protocol. 80 μg protein was denatured for 30 s in 200 μl of 6 m urea at 95 °C. The solution was cooled rapidly to 30 °C, and 800 μl of digest buffer containing trypsin was added immediately. Digest buffer consisted of 50 mm HEPES (pH 8.0), 300 mm NaCl, and 20 mm CaCl2. Trypsin (Sigma T-1426, TCPK-treated bovine) was added to the buffer immediately before use in a 1:2 (w:w) trypsin:GFP ratio. The digest was incubated for 3 min at 30 °C, dithiothreitol was added to a final concentration of 10 mm, and the sample was incubated for 2 min at room temperature. Tryptic peptides were separated by reverse-phase HPLC (Waters 600 HPLC system equipped with a Waters 996 photodiode array detector) on a C18 analytical column (Vydac) using a water/acetonitrile gradient containing 0.05% trifluoroacetic acid. Peptide elution was monitored by absorbance at 220, 280, and 380 nm. Collected fractions were lyophilized immediately.MALDI Mass Spectrometry—Lyophilized peptides were resolubilized in 20 μl of 50% acetonitrile, 50% water, and 0.1% trifluoroacetic acid. 1-μl peptide solution was mixed with 2 μl of a saturated solution of α-cyano-4-hydroxycinnamic acid matrix dissolved in the same solvent system. 1 μl of this preparation was dried on a stainless steel sample plate for data acquisition. Data were obtained using a Voyager DE STR mass spectrometer equipped with a nitrogen laser that produced 337-nm pulses of a 3-ns duration with a repetition rate of 20 Hz. Mass spectra were acquired in the positive ion mode using delayed extraction and the reflectron mode. Each mass spectrum was the average of at least 100 laser shots. Calibration was performed using CalMix 2 (Applied Biosystems) as an external standard, which consists of a mixture of polypeptides ranging in mass from 1,296 to 3,658 Da. Monoisotopic masses obtained for the calibration peptides were accurate within 0.1 Da over the mass range examined. For every EGFP variant assayed, all of the major 220-nm absorbing HPLC peaks were subjected to MALDI mass spectrometry. Reported masses are those that correlate with theoretically possible tryptic peptides containing residues 65–67 (Table II). The peptide identity was verified by N-terminal amino acid sequencing.Table IIPrinciple peaks observed in MALDI spectra of tryptic peptides containing residues 65–67Peptide sequenceTheoreticalObservedMass lossaAn observed mass loss of 20 Da is consistent with the peptide bearing a mature chromophore (cyclization, oxidation, and dehydration). A mass gain of 2 Da is consistent with the sodium adduct of the chromophore-bearing peptide (-20 + 22 Da). A mass loss of 2 Da is consistent with oxidation (dehydrogenation). Residues 65-67 are highlighted.Interpretationm/zDaEGFP (∼100% mature)LPVPWPTLVTTLTYGVQCFSR2378.262358.24-20.02MatureFICTTGKLPVPWPTLVTTLTYGVQCFSR3128.633108.67-19.96MatureEGFP (∼0% mature)LPVPWPTLVTTLTYGVQCFSR2378.262378.530.27PrecyclizationFICTTGKLPVPWPTLVTTLTYGVQCFSR3128.633129.030.40PrecyclizationLPVPWPTLVTTLTYGVQCFSR2378.262378.470.21PrecyclizationFICTTGKLPVPWPTLVTTLTYGVQCFSR3128.633128.970.34PrecyclizationE222Q (∼30% mature)LPVPWPTLVTTLTYGVQCFSR2378.262357.88-20.38MatureFICTTGKLPVPWPTLVTTLTYGVQCFSR3128.633108.15-20.48MatureLPVPWPTLVTTLTYGVQCFSR2378.262378.06-0.2PrecyclizationFICTTGKLPVPWPTLVTTLTYGVQCFSR3128.633128.4-0.23PrecyclizationE222Q (∼90% mature)LPVPWPTLVTTLTYGVQCFSR2378.262357.77-20.49MatureFICTTGKLPVPWPTLVTTLTYGVQCFSR3128.633108.1-20.53MatureLPVPWPTLVTTLTYGVQCFSR2378.262358.06-20.20MatureFICTTGKLPVPWPTLVTTLTYGVQCFSR3128.633128.28-0.35PrecyclizationLPVPWPTLVTTLTYGVQCFSR2378.282378.22-0.06PrecyclizationFICTTGKLPVPWPTLVTTLTYGVQCFSR3128.633128.58-0.05PrecyclizationR96M (∼0% mature)LPVPWPTLVTTLTYGVQCFSR2378.262378.12-0.14PrecyclizationFICTTGKLPVPWPTLVTTLTYGVQCFSR3128.633128.47-0.16PrecyclizationLPVPWPTLVTTLTYGVQCF2135.122157.0321.91Mature, Na+ adductLPVPWPTLVTTLTYGVQCFSR2378.262378.21-0.05PrecyclizationControl: Y66L (colorless)LPVPWPTLVTTLTLGVQCFSR2328.282326.10-2.18Cyclized and oxidizedFICTTGKLPVPWPTLVTTLTLGVQCFSR3078.653076.44-2.21Cyclized and oxidizeda An observed mass loss of 20 Da is consistent with the peptide bearing a mature chromophore (cyclization, oxidation, and dehydration). A mass gain of 2 Da is consistent with the sodium adduct of the chromophore-bearing peptide (-20 + 22 Da). A mass loss of 2 Da is consistent with oxidation (dehydrogenation). Residues 65-67 are highlighted. Open table in a new tab RESULTSThe highly conserved residues Arg96 and Glu222 are buried in the interior of the protein in close vicinity to the heterocyclic end of the chromophore. To examine the role of these residues in catalyzing chromophore biosynthesis, we have carried out site-directed mutagenesis to generate the single-substitution variants R96M and E222Q relative to EGFP. Each of these variants exhibits the two characteristic UV-visible absorbance bands associated with the mature GFP chromophore (Fig. 1A). The maximum around 400 nm is attributed to the neutral form of the chromophore, and the maximum around 480 nm is attributed to the anionic form (Scheme 1) (3Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar, 14Elsliger M.-A. Wachter R.M. Hanson G.T. Kallio K. Remington S.J. Biochemistry. 1999; 38: 5296-5301Crossref PubMed Scopus (288) Google Scholar, 25Bell A.F. He X. Wachter R.M. Tonge P.J. Biochemistry. 2000; 39: 4423-4431Crossref PubMed Scopus (156) Google Scholar). The chromophore pKa value in EGFP has been reported to be 6.0 (3Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar), whereas the pKa values for E222Q and R96M were estimated from the absorbance scans to be ∼5 and 8. The expression of R96M and E222Q yielded large quantities of highly soluble protein that acquired green color more slowly than the EGFP parent clone, allowing for kinetic measurement of chromophore biogenesis in the folded state of the protein. To a first approximation, the kinetic constants reported for these variants refer to the chemical self-modification steps that follow spontaneously upon attainment of the correct three-dimensional structure (Table I).Fig. 1A, UV-visible absorbance spectra of EGFP variants at pH 7.9. Maturation is complete in EGFP and partially complete in the other variants. - - • • - - - • •, EGFP; - - - - - - - -, E222Q; , R96M. B, kinetics of chromophore formation in E222Q as a function of pH at 30 °C. The first time point was collected at 24% chromophore content. C, kinetics of chromophore formation in R96M as a function of pH at 30 °C. The first time point was collected at 0% chromophore content. D, Maturation kinetics of EGFP as a function of pH at 30 °C. Protein folding and chromophore biosynthesis was induced by rapid dilution of urea-denatured immature protein derived from inclusion bodies. Symbols: ▿, pH 6.0; ○, pH 7.0; ▵, pH 8.0; □, pH 9.0; and ⋄, pH 10.0. For B, C, and D, the increase in chromophore anion absorbance was monitored with time, either in the folded state of the protein (B and D) or in the base-denatured state (C). The kinetic data were curve-fitted to a unimolecular rate equation (Equation 1, see “Experimental Procedures”) with Amax, a variable parameter in B and D, and fixed to 100% maturation in C (∼25% protein mature after 2 months).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IObserved pseudo-first-order time constants for the maturation of EGFP and its variantspHEGFPaTime constants (τ = 1/kobs) were determined by computer-fitting the kinetic data to Equation 1.E222QaTime constants (τ = 1/kobs) were determined by computer-fitting the kinetic data to Equation 1.R96MaTime constants (τ = 1/kobs) were determined by computer-fitting the kinetic data to Equation 1.Average (mean ± S.D.)No.bNumber of independent determinations.Average (mean ± S.D.)No.bNumber of independent determinations.Average (mean ± S.D.)No.bNumber of independent determinations.hhh6.0183 (138)311.5 × 103 (0.7 × 103)17.01.28 (0.59)432.4 (15.3)37.51 × 103 (0.27 × 103)18.00.958 (0.132)37.36 (0.88)34.70 × 103 (0.24 × 103)19.01.03 (0.03)30.857 (0.185)34.56 × 103 (0.16 × 103)19.50.287 (0.020)310.01.05 (0.12)30.302 (0.057)34.32 × 103 (0.13 × 103)1a Time constants (τ = 1/kobs) were determined by computer-fitting the kinetic data to Equation 1.b Number of independent determinations. Open table in a new tab The Rate of E222Q Chromophore Biosynthesis Appears to Depend on the Charge State of the Tyr66-Gly67 Peptide Bond— To prepare E222Q precursor protein for kinetic experiments, a rapid protein expression and purification procedure was employed (21Remington S.J. Wachter R.M. Yarbrough D.K. Branchaud B.P. Anderson D.C. Kallio K. Lukyanov K.A. Biochemistry. 2005; 44: 202-212Crossref PubMed Scopus (121) Google Scholar) that yielded protein pools in which only 24–28% population contained the mature chromophore. Aliquots were diluted into appropriate buffers between pH 6 and 10 and incubated at 30 °C. The increase in the 483-nm absorbance band was monitored with time, and the pseudo-first-order time constant τ was extracted from a curve-fit of the data to a first-order rate expression (Equation 1 and Fig. 1B).Each kinetic run was carried out in triplicate using independently prepared protein pools (Table I). The results indicate significant base catalysis of chromophore formation in E222Q with τ ranging from 183 (±138) h at pH 6 to 0.302 (±0.057) h at pH 10, a 600-fold rate enhancement. At pH 7.0, the time constant was 32.4 h, and at pH 8, the time constant was 7.4 h, considerably slower than the 1-h maturation time determined for EGFP (see below). Surprisingly, at pH 9.5 and 10, the time constant for chromophore formation is at least 3-fold faster than in EGFP at the same pH.This result indicates that the rate" @default.
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• W2000435338 title "Base Catalysis of Chromophore Formation in Arg96 and Glu222 Variants of Green Fluorescent Protein" @default.
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