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

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.