In Vitro Attachment of Bilins to Apophycocyanin

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 263, No. 34, Issue of December 5, pp ,1988 Printed in U.S.A. In Vitro Attachment of Bilins to Apophycocyanin 111. PROPERTIES OF THE PHYCOERYTHROBILIN ADDUCT* (Received for publication, February 9,1988) David M. ArcieroSQ, Jerry L. Dallasn, and Alexander N. GlazerSII From the $Department of Microbiology and Immunology, University of California, Berkeley, California and the llgeneral Electric Company, Medical Systems Group, Fremont, California Addition of phycoerythrobilin (PEB) to apophycocyanin at ph 7.0 resulted in covalent adduct formation. The adduct showed absorbance maxima at 575 and 605 nm and fluorescence emission maxima at 582 and 619 nm. Analysis of bilin peptides obtained upon tryptic digestion of the adduct showed residues a-cys-84 and 8-Cys-82 to be the sites of bilin addition. The product of PEB addition at the a-cys-84 site was shown by H NMR analysis to be a dihydrobiliviolinoid peptide-linked pigment differing in structure from that of the naturally occurring PEB-adduct by the presence of a double bond in between C2 and C3 of ring A. At the 8-Cys-82 site both a dihydrobiliviolinoid and a PEB adduct were obtained. Biliverdin also formed a covalent adduct with apophycocyanin with a X, of 669 nm. These results show that the spontaneous in vitro addition of bilins to apophycocyanin does not exhibit the site selectivity of bilin addition observed in vivo. This offers the opportunity to form novel semisynthetic phycobiliproteins. Numerous cyanobacterial and red algal strains produce several phycobiliproteins with chemically different bilin prosthetic groups. For example the red alga Porphyridium crwnturn contains an R-phycocyanin which carries a phycocyanobilin at a-cys-84, and a phycocyanobilin and a phycoerythrobilin (PEB) at positions a-cys-82 and /3-Cys-153 (1) (Cphycocyanin residue numbering; see Ref. 2). In the B-phycoerythrin produced in the same organism, the homologous cysteine residues carry exclusively PEB groups (3). The amino acid sequence about such corresponding bilin attachment sites is highly conserved irrespective of the chemical nature of the bilin (2,3-5). Consequently, it can be inferred that apoprotein conformation must play a role in determining which bilin is present at a given attachment site. A major incentive for the study of the in vitro addition of bilins to apophycocyanin was to examine the ability of the * This research was supported by the National Institute of General Medical Sciences, Department of Health and Human Services Grant GM28994, and National Science Foundation Grant DMB The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Present address: Dept. of Genetics and Cell Biology, University of Minnesota, St. Paul, MN To whom correspondence should be addressed. The abbreviations used are: PEB, phycoerythrobilin; DBV, 3 - substituted 31,3*,15,16-tetrahydrobiliverdin (3 substituted dihydrobiliviolin); HPLC, high performance liquid chromatography; NOESY, nuclear Overhauser enhancement spectroscopy. apoprotein to discriminate between different bilins. In a preliminary survey (6), we established that apophycocyanin bound and reacted covalently with not only phycocyanobilin but also biliverdin and PEB. The sites of reaction between PEB and apophycocyanin are determined in this study as is the structure of the adducts. The results show that phycocy- anobilin and PEB react at the same sites and, as found earlier for phycocyanobilin (7), the major products of PEB addition differ from the naturally occurring bilin peptides by the presence of an additional double bond in ring A. EXPERIMENTAL PROCEDURES Synechococcus sp. PCC7002 (Agmenellum quudruplicatum) apophycocyanin was purified as previously described (6). Biliverdin was obtained from Sigma. PEB (Fig. 1A) was cleaved from Porphyridium cruentum b-phycoerythrin (8) in the same manner as described in Ref. 6 for phycocyanobilin. Purification of the cleaved bilin by HPLC yielded two major products representing >80% of the total pigment applied to the column. These two components were spectroscopically indistinguishable from each other and from the reported spectrum of synthetic PEB (9). The extinction coefficient for phycoerythrobilin dimethyl ester of 25.2mM cm at 594 nm (11) was used to quantitate PEB in 5% (v/v) HC1 in methanol. Approximately 12 pmol of PEB were obtained from 210 mg of b-phycoerythrin. Apophycocyanin-PEB adducts were prepared by the procedure described for the preparation of phycocyanobilin adducts (6). The same products were obtained when unpurified PEB was used for adduct formation and when the reaction was performed with either of the two major components obtained by purification of PEB by HPLC. The adduct prepared from the reaction of 205 mg of apophycocyanin with PEB was used for the characterization of bilin attachment sites and determination of the structures of the adducts. Trypsin digestion of the apophycocyanin-peb adduct and purification of the resulting bilin peptides followed the procedures described in Ref. 6 for the purification of the peptides from the apophycocyaninphycocyanobilin adduct with the following differences. Initial fractionation of the tryptic digest was performed bygel filtration on Sephadex G-50 in 30% (v/v) aqueous acetic acid. Approximately 25% of the Asmn,-absorbing material eluted in the void volume of the column (Fraction I). This fraction was subdigested with thermolysin (12) and the resulting bilin peptides purified by HPLC. The remaining 75% of the ASm,,-absorbing material eluted near the V, for the column (Fraction 11). The bilin peptides in Fraction I1 were purified by preparative HPLC using the HPLC equipment and gradient system described in Ref. 6. Samples for NMR studies were prepared in the manner described in Ref. 7 except that the final solvent was 10 mm trifluoroacetic acid in 99.96% D,O. All other procedures and instrumentation have been described previously (6, 7). * Similar HPLC chromatography results were reported earlier by Fu et al. (10) for both phycocyanobilin and PEB obtained from C- phycocyanin and phycoerythrin, respectively, by methanol cleavage. Fu et al. (10) concluded from mass spectrometric studies that in each case the two components represent cis-trans isomers of the bile pigment

2 Apophycocyanin-Phycoerythrobilin Adduct A -wc coo- I.6 I I I 562 nm 1 aoc coo- FIG. 1. Structures of (A) phycoerythrobilin, (B) dihydrobiliviolin, (C) tryptic PEB peptides from phycoerythrins, and (D) tryptic peptides from the apophycocyanin-peb adduct. For peptides derived from the a-1 site, R = Cys*-Ala-Arg, and for peptides from the 8-1 site, R = Met-Ala-Ala-Cys*-Leu-Arg. Cys* is the residue to which the bilin is attached through a thioether linkage. u W z a 0.8 w m a C WAVELENGTH (nm) FIG. 3. Absorption spectrum of peptide 8-1 DBV in 10 mm trifluoroacetic acid. WAVELENGTH (nm) FIG. 2. Absorption spectrum of the apophycocyanin-peb adduct in 0.06 M sodium phosphate buffer, ph 7.0. RESULTS AND DISCUSSION Preparation and Characterization of the Apophycocyanin- PEB Adduct-The qualitative aspects of the reaction of PEB with apophycocyanin paralleled closely those described for the interaction of phycocyanobilin with the apoprotein (6). The interaction results in a substantial increase in the absorbance of the bilin in the red region of the spectrum as well as a decrease in the near-uv region. Both a fast phase and a slow phase to the absorbance change is evident. A striking observation is that upon adduct formation two long wavelength absorption maxima are seen, one at 575 nm and the other at 605 nm (Fig. 2). The adduct is fluorescent. Excitation at 530 nm leads to fluorescence emission with maxima at 582 and 619 nm. In contrast, the absorption spectrum of b- phycoerythrin shows maxima at 543 and 563 nm (8, 13). Unfolding of the apophycocyanin-peb adduct in 9 M urea at ph 2.5 results in a spectrum with a long wavelength maximum at 560 nm. This spectrum is equivalent to that obtained at acid ph for small bilin peptides obtained by trypsin digestion of the apophycocyanin-peb adduct (Fig. 3). The presence of two distinct long wavelength maxima for the PEB adduct raised the possibility that two different products were being generated as a consequence of heterogeneity in the PEB preparation used for adduct formation. This possibility was ruled out as follows. Analysis of PEB by HPLC before and after reaction with apophycocyanin revealed that two species were disappearing from the pool of crude PEB used in these experiments, the major PEB species and the minor species present at about 15-20% of the major species.2 These two species were therefore isolated individually, reacted with apophycocyanin in separate experiments, and the adducts purified. Both adducts exhibited the same absorption spectra. Tryptic Bilin Peptides Derived from the Apophycocyanin- PEB Adduct-On gel filtration on Sephadex G-50, the bilin peptides in the tryptic digest of the PEB adduct separated into two fractions, I and 11, eluting at Vo and Ve, respectively. Fractionation of the peptides in Fraction I1 by preparative HPLC led to the purification of three major peptides, designated a-1 DBV, 6-1 DBV, and 6-1 PEB, which represented 30, 27, and 17%, respectively, of the total A660,,-absorbing material injected onto the reverse phase column. In addition, 12 minor peptides, ranging in amount from 1 to 7% of the total 560-nm-absorbing material, were resolved on the column as well. The major peptides and most of the minor peptides were subjected to amino acid analysis. One of the major peptides, a-1 DBV,gave the composition Cysl, Alal, Arg,, characteristic of the tryptic peptide that includes the a-cys- 84 residue that functions as the bilin attachment site in the a subunit of C-phycocyanins (14-18). Peptides 6-1 DBV and p-1 PEB and the minor peptides had the composition Metl, Ala2, CYSI, Leul, Arg,, characteristic of the sequence of the tryptic peptide that includes cysteine residue 6-82, one of the two attachment sites on the 6 subunit of C-phycocyanins (14-18). Of the three major bilin peptides, a-1 DBV and p-1 DBV exhibited virtually identical absorption spectra at acid ph with a maximum at 562 nm along with a prominent shoulder on the red edge of the main absorption band that resulted in significant absorbance at 600 nm (Fig. 3). In contrast, the

3 18360 Apophycocyanin-Phycoerythrobilin Adduct 2,7, CHis \r 8,12-CH,CHZCOOH -\ t 10-H l 5 H FIG. 4. The 500-MHz 'H NMR snectrum of the tryptic tripeptide a-1 DBV recorded in 10 mm trifluoroacetic acid in 99.96% D20. PPM absorption spectrum of peptide p-1 PEB showed the characteristics of PEB peptides obtained from phycoerythrins with a maximum at 550 nm and very low absorbance at 600 nm. Tryptic peptides including p-cys-153, the remaining bilin attachment site in C-phycocyanin, were absent from Fraction 11. Since approximately 25% of the 560-nm-absorbing material eluted in Fraction I, the possibility that peptides from this region of C-phycocyanin were present in this fraction was examined by a further digestion of this fraction with thermolysin. The numerous bilin peptides produced by this treatment were purified by HPLC and their amino acid compositions analyzed. No bilin peptides characteristic of the region including p-cys-153were detected. The presence solely of peptides including a-cys-84 and p-cys-82 indicated that Fraction I represented incomplete tryptic digestion products of the apophycocyanin-peb adduct. 'H NMR Analysis-The 'H NMR spectra of peptides a-1 DBV and p-1 DBV recorded in 10 mm trifluoroacetic acid in D20 are shown in Figs. 4 and 5. Chemical shift assignments for the bilin resonances of both peptides are presented in Table I along with previously reported chemical shift assignments for the bilin portions of tryptic PEB peptides encompassing a-cys-84 and p-cys-82 prepared from B- and R- phycoerythrins (12,19). A two-dimensional homonuclear coupled (COSY) analysis was performed on the two DBV peptides (data not shown). Insufficient material precluded a complementary NOESY analysis of the peptides. In the absence of the NOESY analysis, a complete direct assignment of all tetrapyrrolic resonances was not possible. In particular, the singlets observed between 2.14 and 2.26 ppm could not be assigned to particular methyl groups at the 2, 7, 13, and 17 positions. The remaining assignments required the comparison of the two DBV NMR spectra (Figs. 4 and 5) with the previously reported spectra of the corresponding PEB peptides. As is evident from an inspection of Table I, the NMR spectra for the two sets of peptides are quite similar, especially those resonances arising from protons associated with rings B, C, and D of the bilins (see Fig. 1, structures C and D). The only significant deviations can be accounted for by the presence of an additional double bond in the bilin of the DBV peptides at the C2-C3 position of ring A. Thus, resonances observed for the 2-H (2.68 ppm) and 3-H (3.10, 3.22 ppm) in the PEB peptides are missing in the spectra of the two DBV peptides. In addition, there is a significant downfield shift, relative to the PEB peptides, for the 2-CH3 (-0.9 ppm), 3'-H (1.06 and 0.94 ppm), 3'-CH3 (0.37 and 0.34 ppm), and 5-H (0.87 and 0.80 ppm) resonances of the DBV peptides. As expected, the extent of the shift diminishes with increasing distance from the additional site of unsaturation. Evidence that peptides a-1 DBV and p-1 DBV both contain the same bilin is found through a comparison of the chemical shift assignments for the bilin resonances of the two peptides. With the exception of the 3'-H (0.08 ppm), the resonances of all of

4 Apophycocyanin-Phycoerythrobilin Adduct ,7,13,17-CH;s 'r 15-H (1) and Cys P-CH(2) 8,12-CH,CH,COOH 8,12-CHzCHzCOOH 9 a FIG. 5. The 500-MHz 'H NMR spectrum of the tryptic hexapeptide 8-1 DBV recorded in 10 mm trifluoroacetic acid in 99.96% DzO. T- O PPM the other corresponding protons are within 0.04 ppm. This is a higher degree of correlation than that observed in a comparison of the two reference PEB peptides with each other. The abbreviations, a-1 DBV and p-1 DBV, for the two major bilin peptides derived from the adduct were chosen to designate the dihydrobiliviolinoid structure (Fig. 1, B and D) indicated by the NMR analysis for the bilin moieties of these peptides. Properties of the Biliverdin Adduct-Biliverdin reacted with apophycocyanin to form a covalent adduct with distinctive spectroscopic properties. The purified adduct exhibited a long wavelength visible absorption maximum at 669 nm (Fig. 6). Attempts to determine the sites of biliverdin addition by tryptic digestion of the adduct and peptide characterization, as described above for the PEB-adduct, were unsuccessful because of the lability of the biliverdin peptides. Loss of bilin absorbance was encountered.during enzyme digestion and purification. The absorption spectrum of the biliverdin adduct lies further to the red than that of any of the naturally occurring major phycobiliproteins (20). Only the minor phycocyanobilin-containing phycobiliproteins, allophycocyanin B (21), and the high molecular weight polypeptide of the phycobilisome core (22, 23) have absorption maxima at 670 nm. Concluding Remarks-The studies of bilin addition to apophycocyanin lead to several general observations. Binding of the bilin to the apoprotein is a prerequisite to covalent at- tachment (6). Yet, in vitro, the apophycocyanin binding sites do not discriminate between phycocyanobilin and PEB. It is likely that biliverdin also reacts at the same sites. Biliverdin is believed to be the metabolic precursor of phycocyanobilin (24). In vivo, only phycocyanobilin is found attached to C- phycocyanin in wild type cells. These findings provide strong support for the view that in vivo the specificity of bilin attachment is mediated by enzyme catalysis involving appropriate lyases and/or isomerases. Two aspects of the in vitro phycocyanobilin and PEB addition to apophycocyanin remain obscure. In both cases, reaction is limited to a-cys-84 and 8-Cys-82, with a total absence of reaction at the remaining attachment site, p-cys The phycocyanobilin at in C-phycocyanin has been shown to differ in conformation from those at a-84 and The latter have an all-z configuration, whereas in the former ring D is almost perpendicular to ring C (25, 26). This geometry corresponds formally to an E configuration of the 15,16 double bond (27). It is possible that because of this difference in stereochemistry, in the absence of enzyme catalysis, the proper conformers required for binding at are not available in the phycocyanobilin and PEB preparations. A second unexpected feature of the in vitro addition is the oxidative reaction leading to the introduction of an additional double bond between C2 and C3 of ring A both in the phycocyanobilin (6, 7) and the PEB adducts. It is noteworthy that this reaction proceeds to completion for bilins added at the

5 ~ ~ Apophycocyanin-Phycoerythrobilin Adduct TABLE I 'H NMR assignments of the bilin moieties of the a-1 DBV, 0-1 DBV, a-1 PEB, and p-1 PEB bilinpeptides in 10 mm trifluoroacetic acid in DzO at 25 "C Chemical shift, multiplicitf, and JH.H (Hz) Assignment 0-1 DBV a-1 PEB' 2-H - d - d 2.71 (m) 2.67 (m) (3.6, 7.4) 2-C& (s)e (s)e 1.30 (d) (7.4) 1.28 (d) (6.9) 3-H - d - d 3.24 (m) 3.22 (m) (3.0, 3.6) 3"H 4.50 (m) 4.42 (m) ) (m)(3.1, 3.48 (m) (3.0, 6.9) 3"CH (d) (7.1) 1.73 (d) (7.1) 1.40 (d) (7.0) 1.39 (d) (7.4) 5-H 6.73 (8) 6.69 (8) 5.88 (8) 5.89 (s) 7-CH (s)~ (s)~ (9)' (8)' 10-H 7.67 (s) 7.71 (s) 7.38 (5) 7.39 (8) 8,12-CH2CH&OOH 2.77 (m) (t,t) 7.3) (7.2, 2.64 (m) 2.63 (t,t) (7.3, 7.6) 8,12-CH2m&OOH 3.20 (m) 3.22 (m) 3.06 (m) 3.04 (m) 13-CK (s)~ (sy (8)' (8)' 15-H (1) 3.07 (dd)g 3.07 (dd)g ) (dd) (8.4, 3.02 (dd) (8.0, 14.5) 15-H (2) 3.53 (dd) 14.4) 3.52 (4.6, (dd) 14.7) (4.2, 3.32 (dd) 14.5) (4.5, 3.30 (dd) (4.7, 14.5) 16-H 4.61 (dd)g 4.61 (dd)g 4.42 (dd)(4.7, 8.0) 4.49 (dd) (4.7, 8.0) 17-CH (s)~ (sy (8)' (8)' 18"H (dd) (11.9, 18.1) 6.53 (dd) (11.8, ) (dd) (11.7, 17.9) 6.39 (dd) (11.6, 18.0) 18"-Hb 5.97 (dd) (1.8, 18.1) 6.00 (dd) ) (1.1, (dd) 17.9) (1.5, 5.83 (dd) (1.5, 18.0) 18"-H (dd) (1.8, ) (dd) (1.1, 11.8) 5.42 (dd) (1.4, 11.8) 5.40 (dd) (1.5, 11.6) Multiplicity: 8, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet. From Ref. 5. Peptide moiety has the sequence Cys-Tyr-Arg. e From Ref. 12. Not observed. e Resonances of the 2-CH3, 7-CH3, 13-CH3, and 17-CH3 protons are not resolvable. 'Resonances of the 7-CH3, 13-CH3, and 17-CH3 protons are not resolvable. P Overlapping resonances prevent accurate determination of coupling constant. I 278 nrn I new fluorescent molecules with appropriately chosen excitation and emission maxima would provide potentially valuable new compounds for such applications. 383 nm REFERENCES 1. Bryant, D.A., Hixson, C. S., and Glazer, A. N. (1978) J. Biol. Chem. 253, Zuber, H. (1987) in The Light Reactions (Barber, J., ed) pp , Elsevier Scientific Publishing Co., Amsterdam I 3. Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M. (1984) J. Biol. Chem. 259, Sidler, W., Kumpf, B., Rudiger, W., and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, Klotz, A. V., Glazer, A. N. Bishop, J. E., Nagy, J. O., and Rapoport, H. (1986) J. Biol. Chem. 261, Arciero, D. M., Bryant, D. A., and Glazer, A. N. (1988) J. Biol. I j: Chem. 263, Arciero, D. M., Dallas, J., and Glazer, A. N. (1988) J. Biol. Chem. 263, Glazer, A. N., and Hixson, C. S. (1977) J. Biol. Chem. 262, Gossauer, A., and Weller, J.-P. (1978) J. Am. Chem. SOC. 100, Fu; E., Friedman, L., and Siegelman, H. W. (1979) Biochem. J ,l Chapman, D. J., Cole, W. J., and Siegelman, H. W. (1967) J. Am. Chem. SOC. 89, Klotz, A. V., Glazer, A.N., Bishop, J. E., Nagy, J. O., and ) 4d0 3d dO 6dO Rapoport, H. (1986) J. Bwl. Chem. 261, Gantt, E., and Lipschultz, C. A. (1974) Biochemistry 13, WAVELENGTH (nm) ~~ 2966 F ~ 6. ~ Absorptionspectrum. of the apophycocyanin-biliv- 14. Williams, V. P., and Glazer, A. N. (1978) J. Bid. Chem. 253, erdin adduct in 0.05 M sodium phosphate buffer, ph Frank, G., Sidler, W., Widmer, H., and Zuber, H. (1978) Hoppe- Seyler's Z. Physwl. Chem. 359, a-84 site but is partial for bilins added at the p-82 site. No 16. Troxler, R. F., Ehrhardt, M. M., Mason-Brown, A. S., and Offner, ready explanation for this oxidative step is as yet available. G. D. (1981) J. Biol. Chem. 256, ~ i ~ the ~ lability l ~, to add to apophycocyanin bilins chemi- 17. Pilot, T. J., and FOX, J. L. (1984) Proc. Natl. A d. SCi. u. s. A. 81, different from the ligand 'pens the way for the 18. delorimier, R., Bryant, D. A., Porter, R. D,, Liu, W.-y., Jay, E., semisynthesis of new phycobiliproteins. Phycobiliproteins are and Stevens, S. E., Jr. (1984) Proc. Natl. Acad. Sci. U. S. A. widely used as fluorescent tags for cell sorting and cell anal- 81, yses as well as in immunochemistry (28, 29). Preparation of 19. Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport,

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