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.
Wiegand
Georg
G
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Parbel
Axel
A
Seifert
Markus H J
MH
Holak
Tad A
TA
Reuter
Wolfgang
W
D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods; D002942:Circular Dichroism; D003460:Crystallization; D005561:Formates / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D052980:Phycobilins; D045524:Phycobilisomes; D010798:Phycocyanin / Q000737:chemistry* / Q000302:isolation & purification*; D011487:Protein Conformation; D021122:Protein Subunits; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence; D014961:X-Ray Diffraction
D005561:Formates; D010446:Peptide Fragments; D052980:Phycobilins; D045524:Phycobilisomes; D021122:Protein Subunits; C038397:phycoerythrocyanin; C030544:formic acid; D010798:Phycocyanin