Reconstitution of the peridinin-chlorophyll a protein (PCP): evidence for functional flexibility in chlorophyll binding.
The coding regions for the N-domain, and full length peridinin-chlorophyll a apoprotein (full length PCP), were expressed in Escherichia coli. The apoproteins formed inclusion bodies from which the peptides could be released by hot buffer. Both the above constructs were reconstituted by addition of a total pigment extract from native PCP. After purification by ion exchange chromatography, the absorbance, fluorescence excitation and CD spectra resembled those of the native PCP. Energy transfer from peridinin to Chl a was restored and a specific fluorescence activity calculated which was approximately 86% of that of native PCP. Size exclusion analysis and CD spectra showed that the N-domain PCP dimerized on reconstitution. Chl a could be replaced by Chl b, 3-acetyl Chl a, Chl d and Bchl using the N-domain apo protein. The specific fluorescence activity was the same for constructs with Chl a, 3-acetyl Chl a, and Chl d but significantly reduced for those made with Chl b. Reconstitutions with mixtures of chlorophylls were also made with eg Chl b and Chl d and energy transfer from the higher energy Qy band to the lower was demonstrated.
Miller
David J
DJ
Comparative Genomics Centre, James Cook University, Molecular Sciences Building 21, Townsville, QLD, 4711, Australia.
Catmull
Julian
J
Puskeiler
Robert
R
Tweedale
Helen
H
Sharples
Frank P
FP
Hiller
Roger G
RG
D002338:Carotenoids / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002734:Chlorophyll / Q000378:metabolism*; D000077194:Chlorophyll A; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013057:Spectrum Analysis
D015800:Protozoan Proteins; C073227:peridinin chlorophyll-a protein, Dinophyceae; D002734:Chlorophyll; C016040:peridinin; D002338:Carotenoids; D000077194:Chlorophyll A