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SUPPLEMENTARY FIGURE 1. SDS-PAGE analysis of purified Hyp-complexes and
2
maturases. Proteins and complexes were purified from E. coli BL21Star™ (DE3) cells
3
containing plasmid constructs listed in Table I. Gene expression was performed as described
4
in the methods section. Proteins were separated by SDS-PAGE and the gels were stained by
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Coomassie Brilliant Blue. Purified complexes (acrylamide concentration of the gels): A)
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HypC1-StrepII/HoxH-complex (12%/18%); B) HypC1-StrepII/HypD1-complex (12%/18%);
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C) HypC1-StrepII/HypD1/HypE1-complex (12%/18%); D) HypA2/StrepII-HypB2-complex
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(12%/18%); E) HypC2-StrepII/HoxH-complex (12%/18%); F) HypC2-StrepII/HypD2-
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complex (12%/18%); G) HypC2-StrepII/HypD2/HypE2-complex (18%); H) HypA3/StrepII-
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HypB3-complex (18%); I) HypC1-StrepII/HypD2-complex (18%). 25 µg cell-free extract or
11
4 µg (A, B, D–H) to 6 µg (C, I) of purified complexes were applied per lane. Fragments of
12
gels were composed to highlight relevant lanes. Legend: M = Marker/Protein ladder; CFE =
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Cell-free soluble extract; E = Elution fraction; C = contaminant.
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SUPPLEMENTARY FIGURE 2. Sequence alignment of related HoxW-proteins. The
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conserved nickel binding motif present in all HoxW protein sequences is highlighted in green.
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Cysteines with the capacity to coordinate a putative [4Fe-4S]-cluster are highlighted in red, as
18
are the cysteines in the other sequences at the same position. Notably, two homologues (from
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Cn and Mv) contain a complete CxxCxxC motif. A fourth cysteine positioned outside the
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CxxCxxC-motif is highlighted in yellow (in HoxW: residue no. 49). It must be stressed that a
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putative fourth coordinating residue could be taken by a number of amino acids other than
22
cysteine. Legend: Av = Azotobacter vinelandii HoxW; Gh = Grimontia hollisae HoxW; Mv =
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Mycobacterium vanbaalenii HoxW; Ro = Rhodococcus opacus HoxW; Cn = Cupriavidus
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necator HoxW; Rm = Ralstonia metallidurans HoxW; Nitr = Nitrosospira sp. APG3 HoxW;
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Mc = Methylococcus capsulatus HoxW; Syn = Synechococcus sp. PCC 7002 HoxW; Lm =
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Lyngbya majuscula HoxW.
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SUPPLEMENTARY FIGURE 3. Analysis of the purified HypCDE-complex. The complex
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was purified from E. coli BL21Star™ (pE-[3’]hypC1[wt]hypD1[wt]hypE1) cells subjected to
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autoinduction according to the procedure described in the methods section of the article. A)
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Purification of the ternary complex by affinity chromatography, visually followed by SDS-
32
PAGE on 12/18%-gels stained by Coomassie Brilliant Blue. The two weak bands between
33
30–40 kDa are contaminants associated with HypE1, which were also present upon individual
34
purification of StrepII-HypE1. 25 µg of cell-free extract and 6 µg of purified complex were
35
applied. Legend: M = SDS-PAGE marker/protein ladder; CFE = cell-free soluble extract; E =
36
elution fraction. B) Native PAGE (N-PAGE) and Western-Blotting (WB) of the purified
37
complex. N-PAGE was performed on 4–15% gradient gels. 6 µg of purified complex were
38
applied for the Coomassie-stained gel, whereas 0.5 µg were used for subsequent WB and
39
chemiluminescence detection. Legend: M = N-PAGE marker/protein ladder; E = elution
40
fraction (Coomassie-stained); B = elution fraction (WB-visualization). Fragments of gels in
41
A) and B) were composed to highlight relevant lanes. C) Molecular mass determination of
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HypC1D1E1-species by analytical size-exclusion chromatography. Molecular masses were
43
plotted on a logarithmic scale. Standards and samples were analyzed on a Superdex 200 HR
44
10/300 gel filtration column. Buffers and standards used are described in the methods section
45
of the article. A linear fit with the formula K = –0.14·ln(Mr) + 1.22 (R2 = 0.982) was used for
46
molecular mass determination. The calculated masses of the two distinguished species were
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91 kDa (K = 0.601), corresponding to a monomeric HypC1D1E1-complex and 179 kDa (K =
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0.508), which corresponds to a (HypC1D1E1)2-complex. Legend: K = partition coefficient;
49
Mr = relative molecular mass [kDa].