Characterization of a trimeric light-harvesting complex in the diatom Phaeodactylum tricornutum built of FcpA and FcpE proteins.
Fucoxanthin chlorophyll proteins (Fcps), the light-harvesting antennas of heterokont algae, are encoded by a multigene family and are highly similar with respect to their molecular masses as well as to their pigmentation, making it difficult to purify single Fcps. In this study, a hexa-histidine tag was genetically added to the C-terminus of the FcpA protein of the pennate diatom Phaeodactylum tricornutum. A transgenic strain expressing the recombinant His-tagged FcpA protein in addition to the endogenous wild type Fcps was created. This strategy allowed, for the first time, the purification of a specific, stable trimeric Fcp complex. In addition, a pool of various trimeric Fcps was also purified from the wild-type cells using sucrose density gradient ultracentrifugation and gel filtration. In both the His-tagged and the wild-type Fcps, excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated using circular dichroism spectroscopy. Mass spectrometric analyses of the trimeric His-tagged complex indicated that it is composed of FcpA and FcpE polypeptides. It is confirmed here that a trimer is the basic organizational unit of Fcps in P. tricornutum. From circular dichroism spectra, it is proposed that the organization of the pigments on the polypeptide backbone of Fcps is a conserved feature in the case of chlorophyll a/c containing algae.
Joshi-Deo
Jidnyasa
J
Institute of Molecular Biosciences, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.
Schmidt
Matthias
M
Gruber
Ansgar
A
Weisheit
Wolfram
W
Mittag
Maria
M
Kroth
Peter G
PG
Büchel
Claudia
C
D020418:Algal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002734:Chlorophyll / Q000737:chemistry / Q000378:metabolism; D017377:Diatoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D024341:Xanthophylls / Q000737:chemistry / Q000378:metabolism
D020418:Algal Proteins; D045342:Light-Harvesting Protein Complexes; D024341:Xanthophylls; C025164:fucoxanthin; D002734:Chlorophyll