Chaperonin-mediated reconstitution of the phytochrome photoreceptor.
The native phytochrome photoreceptor was purified to homogeneity from etiolated seedlings of oat (Avena sativa L.) and used for renaturation experiments. Light scattering measurements showed that the GroEL molecular chaperone interacts with non-native phytochrome to suppress aggregation of the refolding polypeptide, following its dilution from a chaotrope. The binary complex formed between non-native phytochrome and GroEL was stable and could be isolated by size exclusion chromatography. Discharge of the photoreceptor from GroEL was obtained with 2 mM MgATP, although 6 mM adenosine 5'-O-(3-thiotriphosphate) was also effective. Phytochrome released from GroEL with MgATP was found primarily in the form of 124-kDa monomers, as judged by size exclusion chromatography and nondenaturing gel electrophoresis, although dimers and other oligomeric forms were also observed. The reconstituted dimers, and other oligomeric forms, preferentially cross-reacted with a monoclonal antibody that recognizes native-like epitopes. In vitro folding reactions, using chemically denatured phytochrome, revealed that successful reconstitution of the photoreceptor required the presence of the GroEL chaperonin and MgATP under the conditions tested. Reconstitution in the presence of GroEL yielded phytochrome that could exhibit photoreversibility between the red-light absorbing and far-red absorbing forms.
Grimm
R
R
Institut für Biologie 2, Freiburg, Germany.
Donaldson
G K
GK
van der Vies
S M
SM
Schäfer
E
E
Gatenby
A A
AA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D018834:Chaperonin 60; D018833:Chaperonins; D002523:Edible Grain / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008027:Light; D010786:Photoreceptor Cells / Q000737:chemistry / Q000378:metabolism*; D010834:Phytochrome / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism; D012542:Scattering, Radiation
D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D011506:Proteins; D010834:Phytochrome; D000255:Adenosine Triphosphate; D018833:Chaperonins