Chromophore-Apoprotein Interactions in Synechocystis sp. PCC6803 Phytochrome Cph1
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- 作者：Chung-Mo Park ; Jae-Yoon Shim ; Song-Sook Yang ; Jeong-Gu Kang ; Jeong-Il Kim ; Zigmund Luka ; and Pill-Soon Song
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：May 30, 2000
- 年：2000
- 卷：39
- 期：21
- 页码：6349 - 6356
- 全文大小：117K
- 年卷期：v.39,no.21(May 30, 2000)
- ISSN：1520-4995
文摘
The secondary, tertiary, and quaternary structures of the Synechocystis Cph1 phytochromewere investigated by absorption and circular dichroism spectroscopy, size exclusion chromatography,and limited proteolysis. The Cph1 protein was coexpressed with a bacterial thioredoxin in Escherichiacoli, reconstituted in vitro with tetrapyrrole chromophores, and purified by chitin affinity chromatography.The resultant Cph1 holoproteins were essentially pure and had the specific absorbance ratio (SAR) of0.8-0.9. Circular dichroism spectroscopy and limited proteolysis showed that the chromophore bindinginduced marked conformational changes in the Cph1 protein. The 
-helical content increased to 42-44%in the holoproteins from 37% in the apoprotein. However, no significant difference in the secondarystructure was detected between the Pr and Pfr forms. The tertiary structure of the Cph1 apoprotein appearedto be relatively flexible but became more compact and resistant to tryptic digestion upon chromophorebinding. Interestingly, a small chromopeptide of about 30 kDa was still predominant even after longertryptic digestion. The N-terminal location of this chromopeptide was confirmed by expression in E. coliand in vitro reconstitution with chromophores of the 32.5 kDa N-terminal fragment of the Cph1 protein.This chromopeptide was fully photoreversible with the spectral characteristic similar to that of the full-size Cph1 protein. The Cph1 protein forms dimers through the C-terminal region. These results suggestthat the prokaryotic Cph1 phytochrome shares the structural and conformational characteristics of plantphytochromes, such as the two-domain structure consisting of the relatively compact N-terminal and therelatively flexible C-terminal regions, in addition to the chromophore-induced conformational changes.
-helical content increased to 42-44%in the holoproteins from 37% in the apoprotein. However, no significant difference in the secondarystructure was detected between the Pr and Pfr forms. The tertiary structure of the Cph1 apoprotein appearedto be relatively flexible but became more compact and resistant to tryptic digestion upon chromophorebinding. Interestingly, a small chromopeptide of about 30 kDa was still predominant even after longertryptic digestion. The N-terminal location of this chromopeptide was confirmed by expression in E. coliand in vitro reconstitution with chromophores of the 32.5 kDa N-terminal fragment of the Cph1 protein.This chromopeptide was fully photoreversible with the spectral characteristic similar to that of the full-size Cph1 protein. The Cph1 protein forms dimers through the C-terminal region. These results suggestthat the prokaryotic Cph1 phytochrome shares the structural and conformational characteristics of plantphytochromes, such as the two-domain structure consisting of the relatively compact N-terminal and therelatively flexible C-terminal regions, in addition to the chromophore-induced conformational changes.