A, domain organization of the fungal phytochrome FphA. Dark
gray bar, variable N-terminal extension; PAS, PAS domain;
GAF, GAF domain; and PHY, phytochrome domain (GAF-related
domain) comprising the N-terminal photosensory domain. The C-terminal
regulatory domain consists of histidine kinase domain (HKD) and
response regulator domain (RRD). The following residues either
essential for chromophore binding or coordination as well as for the signal
output were assigned. C195, biliverdin IXα binding residue;
H504, essential for chromophore coordination; H770,
autophosphorylation site; and D1181, trans-phosphorylation site. The
chemical structure of the covalently bound chromophore biliverdin IXα is
shown on the right. B, domain structure of FphA variants.
FphA, full-length protein; ΔFphA, protein without
N-terminal variable extension; FphAN753, photosensory domain;
ΔFphAN753, photosensory domain without N-terminal variable
extension. C, SDS-PAGE analysis of recombinant,
Streptactin-affinity purified holo-FphA variants. Left
panel: 1, holo-FphA; 2, holo-ΔFphA_H770A;
right panel: 1, holo-FphAN753; 2,
holo-ΔFphAN753. The samples were either loaded onto a 7.5% SDS-gel
(left panel) or onto a 10% SDS-gel (right panel), and the
proteins were visualized by staining with Coomassie Brilliant Blue.