Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120.
The phycobilisome (PBS) of Anabaena sp. PCC 7120 was allowed to dissociate into its constituents and the resulting allophycocyanin (AP) fraction was purified. Its reconstitution yielded a complex which according to negative stain electron microscopy and spectral analysis was identical to the native pentacylindrical PBS core domain. Each cylinder of the central tricylindric unit was comprised of four AP (alphabeta)3 disks. Mass analysis using the scanning transmission electron microscope (STEM) showed the presence of 16 AP trimers in the intact reconstitute, which had a total mass of 1966(+/-66) kDa. Composition analysis indicated an AP trimer distribution of (AP-II):(AP-LCM):(AP-B):(AP-I)=6:2:2:6, i.e. an addition of two AP-I and two AP-II complexes compared to a tricylindrical PBS core domain. Therefore, we suggest that each supplementary half-core cylinder found in pentacylindrical AP core domains is comprised of one AP-I and one AP-II trimer, in agreement with the current model. The structural significance of the 127 kDa core membrane linker polypeptide was further investigated by subjecting the AP core reconstitute to mild chymotryptic degradation. After isolation, the digested complex exhibited a tricylindrical appearance while STEM mass analysis confirmed the presence of only 12 AP complexes. Polypeptide analysis by SDS-PAGE and Edman degradation related the half-cylinder loss to cleavage of the Rep4 domain of the core membrane linker polypeptide. On the basis of these data, a general model for the assembly of the three hemidiscoidal PBS types known to date is discussed.
Ducret
A
A
Institute for Molecular Biology and Biophysics, Federal Institute of Technology, Zürich, CH-8093, Switzerland.
Müller
S A
SA
Goldie
K N
KN
Hefti
A
A
Sidler
W A
WA
Zuber
H
H
Engel
A
A
D000595:Amino Acid Sequence; D017033:Anabaena / Q000737:chemistry*; D002918:Chymotrypsin; D013058:Mass Spectrometry; D017348:Microscopy, Electron, Scanning Transmission; D008969:Molecular Sequence Data; D045524:Phycobilisomes; D010798:Phycocyanin / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure
D045524:Phycobilisomes; C002497:allophycocyanin; D010798:Phycocyanin; D002918:Chymotrypsin