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. 2005 Nov 8;392(Pt 1):103–116. doi: 10.1042/BJ20050826

Figure 6. Requirements of the PLD Cys and the GAF His for bilin binding by D. radiodurans BphP, and BphP1 and BphP2 from Ag. tumefaciens.

Figure 6

Codons for the Cys and His residues were converted into those for the indicated amino acids by site-directed mutagenesis of DrBphP(N321), AtBphP1(N320) and AtBphP2(N505). The recombinant wild-type (WT) and mutant polypeptides were incubated with BV IXα for 1 h and purified by nickel-chelate chromatography. (A) Samples were subjected to SDS/PAGE and either assayed for the bound bilin by zinc-induced fluorescence (Zn) or stained for protein with Coomassie Blue (Prot). Apo, apoprotein prior to BV IXα incubation. (B) Absorption spectra of the samples following an extended incubation in darkness. The absorption maxima of the Pr (DrBphP and AtBphP1) and Pfr (AtBphP2) forms are indicated. The absorption spectra were adjusted for an equal amount of protein as determined by absorbance at 280 nm.