Evolutionary novelty in gravity sensing through horizontal gene transfer and high-order protein assembly
Fig 3
The Phycomyces OCTIN crystal lattice is stabilized by intermolecular disulphide bonds.
(A) Conservation of cysteine residue position shown by sequence alignment. Positions exhibiting clade-specific conservation are shown in the color of the species to which they correspond. The positions of other cysteine residues are shown in black. Connected lines above the human FGE represent cysteine pairs that form disulphide bonds. The arrowhead indicates the catalytic cysteine pair. The FGE domain is indicated by the horizontal black bar. (B) Crystal-enriched fraction separated by SDS-PAGE in the presence (+) and absence (−) of the reducing agent 2-ME. P55, but not p14, migrates as a high-molecular–weight smear in the absence of 2-ME. Note that p46 also shifts in the absence of 2-ME, suggesting that it is a processing variant of p55. (C) Stills taken from a video recording the disassembly of Phycomyces OCTIN crystals by the reducing agent DTT (S1 Movie). (D) Synergistic disassembly of Phycomyces OCTIN crystals by SDS and DTT. While SDS alone is sufficient to completely shift p14 to the supernatant after centrifugation at 100,000 x g, only the combination of SDS and DTT has the same effect on p55. 2-ME, 2-Mercaptoethanol; DTT, dithiothreitol; FGE, formylglycine-generating enzyme; OCTIN, octahedral crystal matrix protein; P, pellet; S, supernatant; SDS, sodium dodecyl sulfate; T, total.