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Purification, crystallization, NMR spectroscopy and biochemical analyses of alpha-phycoerythrocyanin peptides

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons78880

Wiegand,  G.
Huber, Robert / Structure Research, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78692

Seifert,  M. H. J.
Holak, Tad / NMR Spectroscopy, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78123

Holak,  T. A.
Holak, Tad / NMR Spectroscopy, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78563

Reuter,  W.
Huber, Robert / Structure Research, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Wiegand, G., Parbel, A., Seifert, M. H. J., Holak, T. A., & Reuter, W. (2002). Purification, crystallization, NMR spectroscopy and biochemical analyses of alpha-phycoerythrocyanin peptides. European Journal of Biochemistry, 269(20), 5046-5055.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0010-6E30-4
Abstract
The alpha-phycoerythrocyanin subunits of the different phycoerythrocyanin complexes of the phycobilisomes from the cyanobacterium Mastigocladus laminosus perform a remarkable photochemistry. Similar to phytochromes - the photoreceptors of higher plants - the spectral properties of the molecule reversibly change according to the irradiation wavelength. To enable extensive analyses, the protein has been produced at high yield by improving purification protocols. As a result, several comparative studies on the Z - and E -configurations of the intact alpha-subunit, and also on photoactive peptides originating from nonspecific degradations of the chromoprotein, were possible. The analyses comprise absorbance, fluorescence and CD spectroscopy, crystallization, preliminary X-ray measurements, mass spectrometry, N-terminal amino acid sequencing and 1D NMR spectroscopy. Intact alpha- phycoerythrocyanin aggregates significantly, due to hydrophobic interactions between the two N-terminal helices. Removal of these helices reduces the aggregation but also destabilizes the protein fold. The complete subunit could be crystallized in its E -configuration, but the X-ray measurement conditions must be improved. Nevertheless, NMR spectroscopy on a soluble photoactive peptide presents the first insight into the complex chromophore protein interactions that are dependent on the light induced state. The chromophore environment in the Z - configuration is rigid whereas other regions of the protein are more flexible. In contrast, the E -configuration has a mobile chromophore, especially the pyrrole ring D, while other regions of the protein rigidified compared to the Z -configuration.