Abstract:
: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.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Chung YH,Masuda S,Bauer CEdoi
10.1016/S0076-6879(06)22009-3subject
Has Abstractpub_date
2007-01-01 00:00:00pages
184-9eissn
0076-6879issn
1557-7988pii
S0076-6879(06)22009-3journal_volume
422pub_type
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