Design and expression of cysteine-bearing hydrophobic polypeptides and their self-assembling properties with bacteriochlorophyll a derivatives as a mimic of bacterial photosynthetic antenna complexes. Effect of steric confinement and orientation of the polypeptides on the pigment/polypeptide assembly process.
A series of cysteine-bearing hydrophobic polypeptides analogous to a light-harvesting one betapolypeptide (LH1beta) from the LH1 complex from the purple photosynthetic bacterium, Rhodobacter sphaeroides, was synthesized using an Escherichia coli expression system. The cysteine was placed in the C- or N-terminal regions of the polypeptide to investigate the influence of steric confinement and orientation of the polypeptides via disulfide linkages as they were self-assembled with zinc-substituted bacteriochlorophyll a ([Zn]-BChl a). The polypeptides were expressed as water-soluble fusion proteins with maltose-binding protein (MBP). The fusion proteins formed a subunit-type complex with the [Zn]-BChl a in an n-octyl-beta-d-glucopyranoside (OG) micellar solution regardless of the cross-links or the cleavage of the cysteines, judging from absorption, CD, and fluorescence spectra. Following treatment with trypsin, the polypeptides were detached from the MBP portion. Such trypsin-digested polypeptides formed a subunit-type LH complex at 25 degrees C, which also showed that the disulfide linkage was not crucial for the subunit formation. When a polypeptide having cysteine on the C-terminus was assembled at 4 degrees C, the Qy absorption band was remarkably red-shifted to approximately 836 nm, suggesting that the cleavage of the large MBP portion liberates the polypeptides to form the progressive type of complex similar to LH1-type complex. The trypsin-treated polypeptides bearing cysteines in both terminal regions, which are randomly cross-linked, did not form the LH1-type complex under oxidative conditions but did form the complex under reductive conditions. This observation suggests that the polypeptide orientation strongly influences the LH1-type complex formation. The progressive assembly from the subunit to the holo-LH1-type complex following cleavage of MBP portion in a lipid bilayer is also briefly discussed.
Dewa
Takehisa
T
Materials Science and Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.
Yamada
Taku
T
Ogawa
Makiko
M
Sugimoto
Miku
M
Mizuno
Taeko
T
Yoshida
Kiyotaka
K
Nakao
Yoshiaki
Y
Kondo
Masaharu
M
Iida
Kouji
K
Yamashita
Keiji
K
Tanaka
Toshiki
T
Nango
Mamoru
M
D000595:Amino Acid Sequence; D025541:Bacteriochlorophyll A / Q000096:biosynthesis / Q000737:chemistry*; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D045342:Light-Harvesting Protein Complexes / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D025541:Bacteriochlorophyll A; D004269:DNA, Bacterial; D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D010455:Peptides; D011993:Recombinant Fusion Proteins; D003545:Cysteine