Differential interactions of phytochrome A (Pr vs. Pfr) with monoclonal antibodies probed by a surface plasmon resonance technique.
Phytochromes are red- and far-red light-reversible photoreceptors for photomorphogenesis in plants. Phytochrome A is a dimeric chromopeptide that mediates very low fluence and high irradiance responses. To analyze the surface properties of phytochrome A (phyA), the epitopes of 21 anti-phyA monoclonal antibodies were determined by variously engineered recombinant phyA proteins and the dissociation constants of seven anti-phyA monoclonal antibodies with phyA were measured using a surface plasmon resonance (SPR)-based resonant mirror biosensor (IAsys). Purified oat phyA was immobilized on the sensor surface using a carboxymethyl dextran cuvette in advance, and the interactions of each chosen monoclonal antibody against phyA in either red light absorbing form (Pr) or far-red light absorbing form (Pfr) at different concentrations were monitored. The binding profiles were analyzed using the FAST Fit program of IAsys. The resultant values of dissociation constants clearly demonstrated the differential affinities between the phyA epitopes and the monoclonal antibodies dependent upon Pr vs. Pfr conformations. Monoclonal antibody mAP20 preferentially recognized the epitope at amino acids 653-731 in the Pr form, whereas mAA02, mAP21 and mAR07/mAR08 displayed preferential affinities for the Pfr's surfaces at epitopes 494-601 (the hinge region between the N- and C-terminal domains), 601-653 (hinge in PASI domain), and 772-1128 (C-terminal domain), respectively. The N-terminal extension (1-74) was not recognized by mAP09 and mAP15, suggesting that the N-terminal extreme is not exposed in the native conformation of phyA. On the other hand, the C-terminal domain becomes apparently exposed on Pr-to-Pfr phototransformation, suggesting an inter-domain cross-talk. The use of surface plasmon resonance spectroscopy offers a new approach to study the surface properties of phytochromes associated with the photoreversible structural changes, as well as for the study of protein-protein interactions of phytochromes with their interacting proteins involved in light signaling events in plants.
Natori
Chihoko
C
Hitachi Advanced Research Laboratory, Hatoyama, Saitama 350-0395, Japan.
Kim
Jeong-Il
JI
Bhoo
Seong Hee
SH
Han
Yun-Jeong
YJ
Hanzawa
Hiroko
H
Furuya
Masaki
M
Song
Pill-Soon
PS
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D018604:Epitope Mapping; D051942:Phytochrome A / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D051942:Phytochrome A