Open AccessPurification, crystallization, NMR spectroscopy and biochemical analyses of α‐phycoerythrocyanin peptides
Author(s) -
Wiegand Georg,
Parbel Axel,
Seifert Markus H. J.,
Holak Tad A.,
Reuter Wolfgang
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.03221.x
Subject(s) - chromophore , nuclear magnetic resonance spectroscopy , spectroscopy , chemistry , fluorescence spectroscopy , circular dichroism , mass spectrometry , absorbance , phycocyanobilin , phycobilisome , protein subunit , two dimensional nuclear magnetic resonance spectroscopy , crystallography , photochemistry , fluorescence , stereochemistry , biochemistry , cyanobacteria , biology , chromatography , physics , quantum mechanics , phycocyanin , bacteria , gene , genetics
The α‐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 α‐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 α‐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.