A haem cofactor is required for redox and light signalling by the AppA protein of Rhodobacter sphaeroides
Version of Record online: 9 MAY 2007
Volume 64, Issue 4, pages 1090–1104, May 2007
How to Cite
Han, Y., Meyer, M. H. F., Keusgen, M. and Klug, G. (2007), A haem cofactor is required for redox and light signalling by the AppA protein of Rhodobacter sphaeroides. Molecular Microbiology, 64: 1090–1104. doi: 10.1111/j.1365-2958.2007.05724.x
- Issue online: 9 MAY 2007
- Version of Record online: 9 MAY 2007
- Accepted 31 March, 2007.
Figure S2. Titration of haem binding to His-AppAΔN and variants (17 nM). Haemin was added in different concentrations to isolated protein. The mol ratios between the proteins and haemin as indicated below the figure are 1:1 (thick line), 1:2.5 (thin line) and 1:5.5 (dotted line) respectively. The unbound haemin was removed by dialysis. Spectra were run for His-AppAΔN wild type, His-AppAΔN variants and for a control sample of lysozyme (17 nM); data presented are the difference spectra His-AppAΔN minus lysozyme.
Figure S3. Concentration dependency curves of the SPRâ€“ based protein interaction analysis. (A) Interaction of PpsR (100 μg ml-1) with variable amount ofAppAΔN in the presence of 1 μg ml-1 haem (filled triangles) or without haem (open diamonds). (B) Interaction of BLUF (200 μg ml-1) with variable amount of AppAΔN in the presence of 1 μg ml-1 haem (filled triangles) or without haem (open diamonds). (C) Interaction of AppAΔN (200 μg ml-1) with variable amount of BLUF in the presence of 1 μg ml-1 haem and light illumination.
Table S1. Redox- and light-dependent puf and puc expression and BChl contents in strains expressing AppA variants. The parental strain APP11 is impaired in the production of both photopigments and structural components of the photosystem because of an insertional inactivation of the chromosomal appA gene. All strains listed in II carry a pRKappARM plasmid with a randomly mutagenized appA gene and all strains listed in III carry a pRKappA plasmid with a directly mutagenized appA gene. The sites of mutation in each AppA variant are shown in the list. Relative bacteriochlorophyll (BChl) concentrations shown represent the mean and standard deviation of three independent measurements of cultures grown at low oxygen tension (pO2 ≤ 3 μM). The puf and puc mRNA expression levels in control strains (I), AppA variants screened after random mutagenesis (II) and AppA variants constructed by site-directed mutagenesis (III) were monitored by Northern blot analysis in the cultures grown in low oxygen (pO2 ≤ 3 μM) at OD660 within 0.7â€“0.9. Redoxand light-dependent puf and puc transcript levels were also monitored by Northern blot analysis except the ones marked by â€˜aâ€™ at the top right corner, which were monitored by semiquantitative RT-PCR. All the expression levels and regulation effects were normalized to the positive control strain APP11(p484-Nco5), for which the expression levels and regulation effects were set as 100%. +++, more than 70%; ++, 30% to 70%; +, less than 30%; â€“, no effect; n.d., no detectable signal by Northern blot analysis.
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