Abstract
Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15=C16 double bond is fixed in either the E or Z configuration and the C14-C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4=C5 double bond is fixed in the Z configuration, and the C5-C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4=C5 Z C5-C6 syn C15=C16 Z C14-C15 anti stereochemistry and that in the Pfr chromophore the C15=C16 double bond has isomerized to the E configuration, whereas the C14-C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5-C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.
Highlights
The Agp[1] apoprotein was assembled with the chromophores as indicated in the first column
All locked chromophores used in the present study were incorporated into the Agp[2] apoprotein. This was shown by absorbance changes that occur after mixing chromophores with the protein (Fig. 6, c– h, and Fig. 8)
Photoconversion of 5Zs-Agp[2] is again The combination of Agp[1] and Agp[2] on the one side and the comparable with that of 5Zs-Agp[1]: irradiation resulted in a blue extended collection of locked chromophores on the other side shift of the absorption maximum from 692 to 675 nm to the for assembly and photoconversion studies revealed a deeper
Summary
Expression and Purification of Agrobacterium Phytochromes Agp[1] and Agp2—An Escherichia coli expression vector termed pAG1 was used for full-length Agp1. pAG1 encodes for the Agp[1] protein with a C-terminal polyhistidine tag for Ni2ϩ affinity purification (8). The vector was modified to encode for a C-terminal His-tagged protein; expression, purification, and assembly were performed as for Agp[1]. A precise concentration of the bilin stock solution was estimated from the absorbance of the diluted solution of an aliquot in MeOH based on the corresponding molecular extinction coefficient (⑀) of each locked chromophore described above. For photoconversion of Pr to Pfr or Pr to Pnr (the nearred-absorbing form), the samples were irradiated with red light from a light-emitting diode (max ϭ 655 nm; half-bandwidth ϭ 40 nm); the light intensity was 135 mol mϪ2 sϪ1 at the position of the cuvette. The initial rate of back-conversion in darkness or under illumination was estimated from the derivative This value was divided by the initial light-induced absorbance change of a dark-assembled sample, i.e. the value that corresponds to the concentration of the photoproduct.
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