Domain interaction in cyanobacterial phytochromes as a prerequisite for spectral integrity.

Two phytochromes, CphA and CphB, from the cyanobacterium Calothrix PCC7601, with similar size (768 and 766 amino acids) and domain structure, were investigated for the essential length of their protein moiety required to maintain the spectral integrity. Both proteins fold into PAS-, GAF-, PHY-, and Histidine-kinase (HK) domains. CphA binds a phycocyanobilin (PCB) chromophore at a "canonical" cysteine within the GAF domain, identically as in plant phytochromes. CphB binds biliverdin IXalpha at cysteine24, positioned in the N-terminal PAS domain. The C-terminally located HK and PHY domains, present in both proteins, were removed subsequently by introducing stop-codons at the corresponding DNA positions. The spectral properties of the resulting proteins were investigated. The full-length proteins absorb at (CphA) 663 and 707 nm (red-, far red-absorbing P (r) and P (fr) forms of phytochromes) and at (CphB) 704 and 750 nm. Removal of the HK domains had no effect on the absorbance maxima of the resulting PAS-GAF-PHY constructs (CphA: 663/707 nm, CphB: 704/750 nm, P (r)/P (fr), respectively). Further deletion of the "PHY" domains caused a blue-shift of the P (r) and P (fr) absorption of CphA (lambda (max): 658/698 nm) and increased the amount of unproperly folded apoprotein, seen by a reduced capability to bind the chromophore in photoconvertible manner. In CphB, however, it practically impaired the formation of P (fr), i.e., showing a very low oscillator strength absorption band, whereas the P (r) form remains unchanged (702 nm). This finding clearly indicates a different interaction between domains in the "typical", PCB binding and in the biliverdin-binding phytochromes, and demonstrates a loss of oscillator strength for the latter, most probably due to a strong conformational distortion of the chromophore in the CphB P (fr) form.