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Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM.

Sonani RR, Gupta GD, Madamwar D, Kumar V - PLoS ONE (2015)

Bottom Line: Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex.A09DM).More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.

View Article: PubMed Central - PubMed

Affiliation: BRD School of Biosciences, Vadtal Road, Satellite Campus, Sardar Patel University, Vallabh Vidyanagar, India.

ABSTRACT
Isolated phycobilisome (PBS) sub-assemblies have been widely subjected to X-ray crystallography analysis to obtain greater insights into the structure-function relationship of this light harvesting complex. Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex. Phycocyanobilin (PCB) chromophores, covalently bound to conserved Cys residues of α- and β- subunits of APC, are responsible for solar energy absorption from phycocyanin and for transfer to photosynthetic apparatus. In the known APC structures, heterodimers of α- and β- subunits (known as αβ monomers) assemble as trimer or hexamer. We here for the first time report the crystal structure of APC isolated from a marine cyanobacterium (Phormidium sp. A09DM). The crystal structure has been refined against all the observed data to the resolution of 2.51 Å to Rwork (Rfree) of 0.158 (0.229) with good stereochemistry of the atomic model. The Phormidium protein exists as a trimer of αβ monomers in solution and in crystal lattice. The overall tertiary structures of α- and β- subunits, and trimeric quaternary fold of the Phormidium protein resemble the other known APC structures. Also, configuration and conformation of the two covalently bound PCB chromophores in the marine APC are same as those observed in fresh water cyanobacteria and marine red algae. More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.

No MeSH data available.


Related in: MedlinePlus

PAGE analysis of the purified allophycocyanin.A) Silver stained and zinc acetate stained 15% SDS-PAGE analyses of purified Phormidium APC. Protein molecular mass standard are shown in lane M. Only two bands were observed on silver stained and zinc acetate stained SDS-PAGE. These correspond to α- and β- subunits of the purified APC, suggesting also absence of linker peptide in purified APC complex. Nearly 10 μg of APC protein was loaded in each lane. B) Silver stained and zinc acetate stained 12% Native-PAGE of purified Phormidium APC further confirm homogeneity of the purified protein.
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pone.0124580.g002: PAGE analysis of the purified allophycocyanin.A) Silver stained and zinc acetate stained 15% SDS-PAGE analyses of purified Phormidium APC. Protein molecular mass standard are shown in lane M. Only two bands were observed on silver stained and zinc acetate stained SDS-PAGE. These correspond to α- and β- subunits of the purified APC, suggesting also absence of linker peptide in purified APC complex. Nearly 10 μg of APC protein was loaded in each lane. B) Silver stained and zinc acetate stained 12% Native-PAGE of purified Phormidium APC further confirm homogeneity of the purified protein.

Mentions: Exponentially growing cell mass (28 days old) was taken for the extraction and purification of APC (Fig 1). Ice crystal formation during successive freeze (-25°C)—thaw (4°C) cycles ruptured the cell wall and facilitated complete extraction of intracellular proteins from cyanobacterial cell. Crude extract was subjected for APC separation using ammonium sulfate precipitation in the presence of Triton X-100. Separated APC was further purified by chromatography techniques. SDS-PAGE profile of purified APC showed only two bands, which corresponded to α- and β- subunits, and substantiated the purity of APC preparation. It also suggested the absence of any linker peptide in purified APC complex (Fig 2A). Emission of orange fluorescence by these bands on zinc acetate stained SDS-PAGE upon UV- illumination indicated the presence of chromophore(s) with both subunits of APC (Fig 2A). Purified APC showed single band in both silver as well as zinc acetate stained native-PAGE (Fig 2B). The functional integrity of purified APC was confirmed by the characteristic emission band observed at around 657 nm upon excitation at 645 nm under fluorescence spectroscope (Fig 3A). UV-visible absorbance spectrum of purified APC showing dominant (653 nm) and shoulder (620 nm) absorption peaks matched well with previously described APC absorption pattern [30,31]. APC specific peaks dominating over the protein specific peak (at 280 nm) in UV-visible spectrum again signified the absence of linker as well as other cellular protein (Fig 3A). Purity ratio, total APC content and total protein content (Table 2) collectively substantiated the success of purification protocol. Peaks at 18056 and 17987 Da were observed in MALDI-TOF experiments that were expected to be due to α- and β- subunits (S1 Fig). The molecular mass of the Phormidium APC oligomer was determined to be 118 kDa based on its elution profile on the molecular sieve column (Fig 3B). Given the observed mass of 36043 Da for an αβ monomer, the Phormidium protein is expected to form trimer in the buffer containing 10 mM Tris-HCl (pH, 8.0) and 100 mM sodium chloride.


Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM.

Sonani RR, Gupta GD, Madamwar D, Kumar V - PLoS ONE (2015)

PAGE analysis of the purified allophycocyanin.A) Silver stained and zinc acetate stained 15% SDS-PAGE analyses of purified Phormidium APC. Protein molecular mass standard are shown in lane M. Only two bands were observed on silver stained and zinc acetate stained SDS-PAGE. These correspond to α- and β- subunits of the purified APC, suggesting also absence of linker peptide in purified APC complex. Nearly 10 μg of APC protein was loaded in each lane. B) Silver stained and zinc acetate stained 12% Native-PAGE of purified Phormidium APC further confirm homogeneity of the purified protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414346&req=5

pone.0124580.g002: PAGE analysis of the purified allophycocyanin.A) Silver stained and zinc acetate stained 15% SDS-PAGE analyses of purified Phormidium APC. Protein molecular mass standard are shown in lane M. Only two bands were observed on silver stained and zinc acetate stained SDS-PAGE. These correspond to α- and β- subunits of the purified APC, suggesting also absence of linker peptide in purified APC complex. Nearly 10 μg of APC protein was loaded in each lane. B) Silver stained and zinc acetate stained 12% Native-PAGE of purified Phormidium APC further confirm homogeneity of the purified protein.
Mentions: Exponentially growing cell mass (28 days old) was taken for the extraction and purification of APC (Fig 1). Ice crystal formation during successive freeze (-25°C)—thaw (4°C) cycles ruptured the cell wall and facilitated complete extraction of intracellular proteins from cyanobacterial cell. Crude extract was subjected for APC separation using ammonium sulfate precipitation in the presence of Triton X-100. Separated APC was further purified by chromatography techniques. SDS-PAGE profile of purified APC showed only two bands, which corresponded to α- and β- subunits, and substantiated the purity of APC preparation. It also suggested the absence of any linker peptide in purified APC complex (Fig 2A). Emission of orange fluorescence by these bands on zinc acetate stained SDS-PAGE upon UV- illumination indicated the presence of chromophore(s) with both subunits of APC (Fig 2A). Purified APC showed single band in both silver as well as zinc acetate stained native-PAGE (Fig 2B). The functional integrity of purified APC was confirmed by the characteristic emission band observed at around 657 nm upon excitation at 645 nm under fluorescence spectroscope (Fig 3A). UV-visible absorbance spectrum of purified APC showing dominant (653 nm) and shoulder (620 nm) absorption peaks matched well with previously described APC absorption pattern [30,31]. APC specific peaks dominating over the protein specific peak (at 280 nm) in UV-visible spectrum again signified the absence of linker as well as other cellular protein (Fig 3A). Purity ratio, total APC content and total protein content (Table 2) collectively substantiated the success of purification protocol. Peaks at 18056 and 17987 Da were observed in MALDI-TOF experiments that were expected to be due to α- and β- subunits (S1 Fig). The molecular mass of the Phormidium APC oligomer was determined to be 118 kDa based on its elution profile on the molecular sieve column (Fig 3B). Given the observed mass of 36043 Da for an αβ monomer, the Phormidium protein is expected to form trimer in the buffer containing 10 mM Tris-HCl (pH, 8.0) and 100 mM sodium chloride.

Bottom Line: Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex.A09DM).More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.

View Article: PubMed Central - PubMed

Affiliation: BRD School of Biosciences, Vadtal Road, Satellite Campus, Sardar Patel University, Vallabh Vidyanagar, India.

ABSTRACT
Isolated phycobilisome (PBS) sub-assemblies have been widely subjected to X-ray crystallography analysis to obtain greater insights into the structure-function relationship of this light harvesting complex. Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex. Phycocyanobilin (PCB) chromophores, covalently bound to conserved Cys residues of α- and β- subunits of APC, are responsible for solar energy absorption from phycocyanin and for transfer to photosynthetic apparatus. In the known APC structures, heterodimers of α- and β- subunits (known as αβ monomers) assemble as trimer or hexamer. We here for the first time report the crystal structure of APC isolated from a marine cyanobacterium (Phormidium sp. A09DM). The crystal structure has been refined against all the observed data to the resolution of 2.51 Å to Rwork (Rfree) of 0.158 (0.229) with good stereochemistry of the atomic model. The Phormidium protein exists as a trimer of αβ monomers in solution and in crystal lattice. The overall tertiary structures of α- and β- subunits, and trimeric quaternary fold of the Phormidium protein resemble the other known APC structures. Also, configuration and conformation of the two covalently bound PCB chromophores in the marine APC are same as those observed in fresh water cyanobacteria and marine red algae. More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.

No MeSH data available.


Related in: MedlinePlus