Purification and reconstitution of PYP-phytochrome with biliverdin and 4-hydroxycinnamic acid.
PYP-phytochrome (Ppr) is a unique photoreceptor that contains a blue light-absorbing photoactive yellow protein (PYP) domain, a red light-absorbing phytochrome domain, and a histidine kinase domain. This chapter describes overexpression of Ppr in a strain of Escherichia coli that allows covalent attachment of substoichiometric amounts of biliverdin in vivo. Ppr is then fully reconstituted with biliverdin, followed by attachment of 4-hydroxycinnamic acid (p-coumaric acid), in vitro. Holo-Ppr with both chromophores is then isolated via an affinity tag and quantified for chromophore attachment by analysis of the absorption spectrum for biliverdin and 4-hydroxycinnamic acid. We also provide conditions for measuring autophosphorylation of Ppr.
Chung
Young-Ho
YH
Department of Biology, Indiana University, Bloomington, Indiana, USA.
Masuda
Shinji
S
Bauer
Carl E
CE
D001059:Apoproteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D001664:Biliverdine / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D020283:Photoreceptors, Microbial / Q000302:isolation & purification* / Q000378:metabolism*; D011422:Propionates; D013053:Spectrophotometry
D001059:Apoproteins; D001426:Bacterial Proteins; D003373:Coumaric Acids; D020283:Photoreceptors, Microbial; C120281:Ppr protein, Rhodospirillum centenum; D011422:Propionates; C495469:p-coumaric acid; D001664:Biliverdine