J. Biol. Chem., Vol. 261, Issue 15, 6797-6805, May, 1986
Phycobiliprotein-bilin linkage diversity. II. Structural studies on A- and D-ring-linked phycoerythrobilins
AV Klotz, AN Glazer, JE Bishop, JO Nagy and H Rapoport
The discovery that the phycocyanobilin group attached to Cys-155 of the
beta subunit of C-phycocyanin is D-ring linked (Bishop, J. E., Lagarias, J.
C., Nagy, J. O., Schoenleber, R. W., Rapoport, H., Klotz, A. V., and
Glazer, A. N. (1986) J. Biol. Chem. 261, 6790-6796) prompted examination of
the linkage mode for phycoerythrobilin (PEB) groups attached at the
corresponding position in other biliproteins. Appropriate small peptides
were obtained by exhaustive enzymatic digestion of Porphyridium cruentum
R-phycocyanin (peptide R-PC beta-2TP PEB) and B-phycoerythrin (peptide B-PE
beta-2TP PEB). These peptides had the following structures R-PC beta-2TP
PEB Gly-Asp-Cys(PEB)-Ser-Ser B-PE beta-2TP PEB Cys(PEB)-Thr-Ser. The
spectroscopic and chemical properties of these peptides were compared with
those of P. cruentum B- phycoerythrin peptide alpha-1 PEB,
Cys(PEB)-Tyr-Arg, in which the bilin is A-ring linked (Schoenleber, R. W.,
Leung, S.-L., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1983) J. Am.
Chem. Soc. 105, 4072- 4076). The PEB groups in peptides R-PC beta-2TP PEB
and B-PE beta-2TP PEB were shown to be D-ring linked on the basis of the
following criteria. Secondary ion mass spectrometry showed the bilins in
these peptides and in alpha-1 PEB to have the same mass. The 18'-CH3,
18'-H, and 15-H resonances in the 1H NMR spectra of R-PC beta-2TP PEB and
B-PE beta-2TP PEB appear significantly upfield from the corresponding
thioether-linked ring A resonances seen in the spectrum of peptide alpha-1
PEB. The CD spectra of the two former peptides showed a strong positive
Cotton effect at 300 nm. Such a Cotton effect is absent from the CD
spectrum of peptide alpha-1 PEB and those of other A-ring-linked PEB
peptides. Refluxing in methanol led to a near-quantitative release of PEB
from alpha-1 PEB but no release from R-PC beta-2TP PEB and less than 20%
release from B-PE beta-2TP PEB. In conjunction with earlier studies, these
results show that distinctive amino acid sequences are found about the
attachment sites for A-ring-linked, D-ring-linked, and dilinked (A- and
D-ring-linked) bilins on the alpha and beta subunits of cyanobacterial and
red algal phycobiliproteins and that the mode of linkage can be correctly
predicted from inspection of the amino acid sequence.
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