THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 253, No 1, Issue of January 10, pp. X-219, 1978

Pm&d in U S.A.

Structural Studies on Phycobiliproteins II. C-PHYCOCYANIN: AMINO ACID SEQUENCE OF THE /3 SUBUNIT. SPECIFIC CLEAVAGE OF THE

(Y SUBUNIT*

(Received for publication, July 26, 1977)

PHILIP FREIDENREICH,$ GERALD S. APELL, AND ALEXANDER N. GLAZER§

From the Department of Biological Chemistry, UCLA School of Medicine, and the Molecular Biology Institute, University of California, Los Angeles, California 90024

Studies of the amino acid sequence of the p subunit of Synechococcus sp. 6301 C-phycocyanin are presented. Se- quences were determined of the NH,-terminal cyanogen bromide peptide (residues 1 to 78), and of the cyanogen bromide peptide (residues 86 to 133), containing the sole free cysteinyl residue of the protein. The overlap between these two peptides and the two other cyanogen bromide peptides bearing the bilin chromophores (Williams, V. P., and Glazer, A. N. (1978) J. Biol. Chem. 253, 202-211) was obtained by cleaving the /3 subunit at the sole cysteinyl residue (at position 108) after reaction with 2-nitro-54hio- cyanobenzoate. The following sequence of the /3 subunit is based on these studies:

Tyr-Phe-hsp-Lys-Ala-Ala-Ala-Ala-Val-Ala 170

The sites of bilin attachment (cysteinyl residues 81 and 152) are indicated by asterisks.

The C-phycocyanins derived from the unrelated cyanobac- teria Synechococcus sp. 6301 and Anabaena uariubilis each contain a single tryptophan residue located in the LY subunit. Treatment of these proteins with 2-(2-nitrophenylsulfenyl)- 3-methyl-3-bromoindolenine led to specific cleavage of the (Y subunits at the tryptophan residue. No other cleavage sites were detected. In each case, a 33-residue peptide, representing the COOH terminus of the o subunit was obtained. This indicates that the tryptophan residue occu-

* This investigation has been supported in part by Grant GM11061 and Training Grant GM 00364 from the National Institute of General Medical Sciences, United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “‘uduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, 200 Beaumont Drive, Wallingford, Pa. 19086. 8 Present address, Department of Bacteriology and Immunology,

University of California, Berkeley, Calif. 94720. To whom inquiries should be addressed.

pies the same position in each (Y subunit sequence. Partial amino acid sequences of the two peptides show a high degree of homology.

The cyanogen bromide cleavage of the p subunit of Synecho- coccus sp. 6301 C-phycocyanin at the 3 methionyl residues produced the expected four peptides. A detailed study of the sequence of two of these peptides, each of which carried a covalently linked phycocyanobilin prosthetic group, has been presented (1). The determination of the sequence of the other two peptides is discussed here. Cleavage of the p subunit at the single free cysteinyl residue (2, 3) was exploited to obtain peptides which permitted ordering of the cyanogen bromide

peptides. The (Y subunit of this C-phycocyanin contains a single

tryptophan residue. Selective cleavage at this residue was explored for two major reasons: first, to provide large peptides valuable in the study of the sequence of the 01 subunit; second, to examine if the invariable presence of at least 1 tryptophan

residue in the amino acid composition of the 01 subunits of various phycocyanins (4) also implied identity in its location in the primary sequences of these polypeptide %chains. The latter question was studied by comparing the cleavage prod- ucts obtained by treating two C-phycocyanins from unrelated cyanobacteria with a reagent which produces selective chemi- cal cleavage at tryptophanyl residues (51.’

RESULTS

The p subunit of Synechococcus sp. 6301 C-phycocyanin contains 3 methionyl residues. Cyanogen bromide cleavage of this polypeptide chain produces the four expected peptides. The composition of these peptides is presented in Table I. The sum of their amino acid compositions coincides well with the amino acid composition determined for the p subunit (1, 6). The proline value for the intact p subunit is 1 residue higher than that obtained from the sum of the proline contents of the

1 The “Experimental Procedures,” Figs. 1 to 7 and 9 to 13, and Tables I to XII are presented as a miniprint supplement immediately following this paper. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No. 77111-1177, cite author(s) and include a check or money order for $4.35 per set of photocopies.

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%N-T?R-

Sequence of the /3 Subunit of C-Phycocyanin

NTCB

1 SH

MET-MET- &S -MET

p- S:B”i;T

ALA -COOH 108 133 170

PEPTIDE NTCB-1

He N-TAR

Cleavaqe after reoct10n wth 2-nltro-5-thlocyanobenzoote

I PEPTI DE NTCB-2

M?TT_h?T-A:; tlPNB-CH-CO-NH-E T z----- 170

ALA-COOH

A hHp

CNBr-2 I

Hk -5

i

Trypsin

CNBr cleovoqe \ CNBr cleavage T-12(114-131) T-15035-144) T-17(165-1701

NTCB-l/CNBr-3’ CNBr-4

ZAP HE GE to7 134 170

ARG LYS -ALA-COOH

213

FIG. 8. Cleavage of the p subunit of Synechococcus sp. phycocyanin at the single free cysteine residue. Peptide NTCB-2 was isolated in pure form (Fig. 5, Fraction II). Peptides T-12, T-15, and T-17 were isolated in nure form after tryptic hydrolysis of peptide NTCB-2, and peptide CNBr-4 after CNBr cleavage of peptide NTCB-2. Peptides CNBr-2 and NTCB/CNBr-3’ (which represents the NH,-terminal region of peptide CNBr-3) were isolated as products of the CNBr cleavage of the fraction containing peptide NTCB- 1 (Fig. 5, Fraction I).

individual peptides. It is possible that the proline value obtained for the intact /3 subunit contains a contribution from co-chromatography of a breakdown product of the bilin chro- mophores or from the bilin-linked cysteinyl residues. The studies on the sequence of peptides CNBr-2 and CNBr-4 are presented in the preceding paper (1). The results presented here deal with the determination of the sequences of peptides CNBr-1 and CNBr-3 and the establishment of the relative positions of the four peptides in the sequence of the p subunit.

Amino Acid Sequence of Peptide CNBr-1 -The sequence of this 78-residue NH,-terminal peptide was established by a combination of automated Edman degradation of the p sub- unit (Table II) and of enzymic hydrolysates of peptide CNBr- 1. The results of the studies on the tryptic peptides are presented in Tables III and IV, and those on the chymotryptic peptides in Table V. These studies led to the sequence pro- posed in Fig. 12.

Amino Acid Sequence of Peptide CNBr-3- This peptide contains the sole free cysteinyl residue of Synechococcus sp. 6301 C-phycocyanin (1). Oxidation of this sulfhydryl group takes place during the cleavage of unmodified phycocyanin with CNBr. Consequently, in the initial gel filtration of the cleavage products, this 48-residue peptide elutes as a disullide- linked dimer near the void volume of a Sephadex G-50 column. The dimerization was exploited in a two-step purification of this peptide by gel filtration. In the initial gel filtration, the bilin-containing peptides, CNBr-2 and CNBr-4, are separated from a peak containing the intact a subunit, peptide CNBr-3, and peptide CNBr-1 (see Ref. 1, Fig. 1). The mixture of the latter components can then be separated, after performic acid oxidation, by a second gel filtration (Fig. 1). The sequence of peptide CNBr-3 was determined by automated Edman degra- dation of the intact peptide, as well as by the determination of the sequences of tryptic and chymotryptic peptides derived from peptide CNBr-3. The studies on peptide CNBr-3 are detailed in Tables VI to IX, and the sequence based on these studies is given in Fig. 13.

Cleavage of the /3 Subunit at the Cysteinyl Residue- Com- parison of the NH,-terminal sequences of the intact /3 subunit and that of peptide CNBr-1 permitted its assignment to the NH, terminus. The absence of homoserine (or its lactone) from peptide CNBr-4 (1) placed it at the COOH terminus. The relative positions of peptides CNBr-2 and CNBr-3 were determined through the availability of a single free sullhydryl group on the /3 subunit of C-phycocyanin. Cleavage at this

residue after reaction with 3-nitro-5thiocyanobenzoate led to the isolation of a 63-residue fragment (peptide NTCB-2;’ Table X), which represented the COOH-terminal portion of peptide CNBr-3 and the entire length of peptide CNBr-4. This conclusion was reached on the basis of the following studies. The amino acid composition of peptide NTCB-2 was in close agreement with that calculated by summing residues 108 to 133 present within peptide CNBr-3 (Fig. 13) with those of CNBr-4 (residues 134 to 170). Peptide CNBr-4 was isolated as the sole bilin chromophore-containing peptide on CNBr cleav- age of peptide NTCBS. Three pure tryptic peptides, T-12, T- 15, and T-17 (see Table X) were isolated from the tryptic digest of peptide NTCB-2. All of these peptides represent segments of peptides CNBr-3 and CNBr-4. No tryptic peptides derived from peptide CNBr-1, or the NH,-terminal portion of CNBr-3, were detected.

The above data indicate that a peptide, NTCB-1, which contains residues 1 to 107 must be present in Fraction I (Fig. 6). Cleavage of the pooled peptides in this fraction with CNBr led to the isolation of the expected peptides: CNBr-2 (Fraction IV, Fig. 7) and the NH,-terminal portion of peptide CNBr-3 (Fraction III, Fig. 7) designated NTCB-l/CNBr-3’. The amino acid composition of the latter fraction corresponded to that of residues 86 to 107 in the sequence of peptide CNBr-3. Deter- mination of the NH,-terminal sequence of peptide NTCB-l/ CNBr-3’ by the dansyl-Edman procedure gave the sequence Glu-Ile/Leu, which corresponds to that of peptide CNBr-3. Fraction II (Fig. 7) contained peptide CNBr-4 in a yield approximately 20% that of peptide CNBr-2 obtained from Fraction IV (Fig. 7). Clearly, the cleavage at the cysteinyl residue was not quantitative. This was anticipated from other studies of this reaction (2). The above results are summarized in Fig. 8.

The amino acid compositions of the COOH-terminal pep- tides of the a subunits of Synechococcus sp. 6301 and A. variabilis C-phycocyanins, obtained by cleavage with BNPS- skatole, are shown in Table XI. Sequence studies on these peptides, tabulated in Tables XI and XII, led to the partial sequences presented in Fig. 14.

2 The abbreviations used are: NTCB and BNPS-SK, peptides obtained by reacting the proteins with 2-nitro&thiocyanobenzoate or 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine, respec- tively; T, tryptic peptides; C, chymotryptic peptides; Th, thermolytic peptides; SP, staphylococcal protease peptides; PCB, phycocyanobi- lin; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.

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214 Sequence of the p Subunit of C-Phycocyanin

Carboxyl-terminal fragment of the a-subunit of Synechococcus sp. 6301 phycocyanin

1 5 10 15 Trp) ~r-Val-Glu-Ala-Leu-Lys-Tyr-Ile-Lys-Ala-Asn-His-Gly-Leu-Gly-Ser-Asp-

------+---s+---,-~~-( )-

20 25 30

( Ser-Arg-Asp-Glu-Ala-Asn-(Ser, Tyr, Ile, Asn, Asn, Ile, Leu, Ala, Leu, Ser)

)( )--L-b-+

Carboxyl-terminal fragment of the a-subunit of Anabaena variabilis phycocyanin

1 Trp) Tyr-Val-Glu-Ala-Leu-Lys-His-Ile-Lys-Ala-Asn-His-Gly-Leu-Ala-Ser-Gln-

- CI BNPS-SK-Z/T-l

-7-I

(Gly-Ala-A%) -Glu- (Ala, Asx, Thr, T$, 30

Ile, Asx)-Ala-Ile-Asx-Ala- (Leu, Ser)

rBNPS-SK-2/T-3a- ----

FIG. 14. Summary of studies on the sequences of the COOH-terminal peptides of the (Y subunits of the C-phycocyanins of Synechococcus sp. 6301 and Anabaena variabilis, released upon cleavage with BNPS-skatole. For explanation of symbols see legend to Fig. 12.

1 10 20 Thr-Phe-Asp-Ala-Phe-Thr-Lys-Val-Val-Ala-Gln-Ala-Asp-Ala-Arg-Gly-Glu-Phe-Leu-Ser-Asp-Ala-Gln-Leu-Asp-Ala-Ser-Leu-Arg-

30 40 50 Leu-Val-Ala-Glu-Gly-Asn-Lys-Arg-Ile-Asp-Thr-Val-Asn-Arg-Ile-Thr-Gly-Asn-Ala-Ser-Ser-Ile-Val-Ala-Asn-Ala-Ala-Arg-Ala-

CNBr-1 1 80 'iB

CNBr-2 60 70 1

Leu-Phe-Ala-Glu-Gln-Pro-Ser-Leu-Ile-Ala-Pro-Gly-Gly-Asn-Ala-Tyr-Thr-Asn-A~g-Met~Ala-Ala-Cys-Leu-Arg-Asp-MetY-Glu-Ile-

NTCB

90 100 I 110 Ile-Leu-Arg-Tyr-Val-Thr-Tyr-Ala-Val-Phe-Thr-Gly-Asp-Als-Ser-Ile-Leu-Asp-Asp-Arg~Cys-Leu-Asp-Gly-Leu-Arg-Glu-Thr-Tyr-

120 130 CNBr-,r-4 140 Leu-Ala-Ser-Gly-Val-Pro-Gly-Ala-Leu-Val-Ala-Glu-Gly-Vsl-Arg-Lys-Met~Lys-Asp-Ala-Ala-Val-Ala-Ile-Val-Ser-Asp-Arg-Asn-

PCB

150 I 160 170 Gly-Ile-Thr-Gln-Gly-Asp-(Cys,Ser)-Ala-I1e-Ser-Glu-Leu-Gly-Ser-Tyr-Phe-Asp-Lys-Ala-Ala-Ala-Ala-Val-Ala

FIG. 15. Amino acid sequence of the /3 subunit of Synechococcas sp. 6301 C-phycocyanin.

DISCUSSION

The sequence of the p subunit of Synechococcus sp. 6301 C- phycocyanin based on the studies presented in this and the preceding paper (1) is shown in Fig. 15. The sequences about the 2 bilin-linked cysteinyl residues are totally distinct. Both sequences are unrelated to that surrounding the free cysteinyl residue 108. It is not clear from these data what features determine the specific residues serving as sites of attachment for the bilin groups. This point is discussed further elsewhere (7).

The pentapeptide sequence Leu-Val-Ala-Glu-Gly occurs twice, at residues 30 to 34 and 125 to 129. However, there is

no evidence of extensive internal homologous regions within the p subunit. Examination of the linear sequence reveals the absence of large hydrophobic or hydrophilic stretches. There is little clustering of charged residues or of hydroxy- amino acids. The linear sequence would permit formation of several helical regions (8), adding up to -40% of cr helix in this subunit.

A single tryptophan residue appears to be a characteristic constituent of the (Y subunits of C-phycocyanins and R-phyco- cyanins (6). It was of interest to determine if specific cleavage of the a: subunit could be obtained at this residue, and, further, if the position of the tryptophan residue was invariant

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Sequence of the p Subunit of C-Phycocyanin 215

in the sequence of the (Y subunits derived from C-phycocyanins of unrelated organisms.

The two C-phycocyanins compared in this study were de- rived from representatives of the two major taxonomic classes of cyanobacteria-Synechococcus sp. 6301, a unicellular cy- anobacterium, and A. variabilis, a filamentous organism. In each instance, the phycocyanin contains a single tryptophan residue, that present in the (Y subunit (6, 9). Cleavage of these proteins with BNPS-skatole was restricted to the peptide bond COOH-terminal to the tryptophyl residue. As illustrated in Fig. 14, a 33-residue COOH-terminal fragment of the (Y subunit is released in each case. (The amino acid sequence of these peptides is very different from the known NH,-terminal sequences of the two (Y subunits (10, ll), thus excluding the possibility that these fragments represent NH,-terminal seg- ments of the (Y subunits.) The results of the BNPS-skatole cleavage indicate that the tryptophyl residue occupies the same position in the amino acid sequence of both proteins. The high degree of homology observed between the partial sequences of the two carboxyl terminal peptides (Fig. 14) parallels that observed for the NH,-terminal sequences of these two polypeptide chains (10).

The homology of the primary sequences of biliproteins derived from phylogenetically distantly related organisms is

further documented by the comparison of the sequences about the bilin binding regions of different biliproteins presented in the accompanying paper (7).

Acknowledgment-We are indebted to Dorothy McNall for the amino acid analyses.

1.

2. 3.

Williams, V. P., and Glazer, A. N. (1978). J. Biol. Chem. 253, 202-211

Degani, Y., and Patchornik, A. (1974) Biochemistry 13, 1-11 Jacobson, G. R., Schaffer, M. H., Stark, G. R., and Vanaman,

T. C. (1973) J. Biol. Chem. 248. 6583-6591 4. Glazer, A. N. (1976) Photochen. Photobiol. Reu. 1, 71-115 5. Omenn. G. S.. Fontana, A., and Anfinsen, C. B. (1970) J. Biol.

Cheni. 245, i895-1902. 6. Glazer, A. N., and Fang, S. (1973) J. Biol. Chem. 248, 659-662 I. Brvant. D. A.. Hixson. C. S.. and Glazer. A. N. (1978) J. BioZ.

8. 9.

i?hei. 253, 220-225 Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13,222-245 Bryant, D. A., Glazer, A. N., and Eiserling, F. A. (1976) Arch.

Microbial. 110, 61-75

REFERENCES

10. Glazer, A. N., Apell, G. S., Hixson, C. S., Bryant, D. A., Rimon. S.. and Brown. D. M. (1976) Proc. N&Z. Acad. Sci. U. S. A. i0, 3428-3431 ’

11. Williams. V. P.. Freidenreich, P.. and Glazer. A. N. (1974) Biochek. Biophys. Res. Comkm: 59, 462-466

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216 Sequence of the p Subunit of C-Phycocyanin

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Sequence of the p Subunit of C-Phycocyanin 217

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218 Sequence of the /3 Subunit of C-Phycocyanin

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P Freidenreich, G S Apell and A N Glazerof the beta subunit. Specific cleavage of the alpha subunit.

Structural studies on phycobiliproteins II. C-phycocyanin: amino acid sequence

1978, 253:212-219.J. Biol. Chem. 

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