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. 2015 Jun 18;290(31):19319–19333. doi: 10.1074/jbc.M115.662379

FIGURE 1.

FIGURE 1.

UV-visible spectroscopy, native PAGE, and DLS analysis of SiR. A, the UV-visible spectrum of recombinant SiR shows the expected peaks at 386, 455, and 590 nm at the expected ratios, characteristic of the overlapping siroheme and flavin cofactors of the SiR holoenzyme (4) (inset). B, 4–16% BisTris NativePAGE gel. Lane 1, NativeMark size standards (in kDa; Life Technologies). SiRHP (HP) and SiRFP (FP) controls are shown in lanes 2 and 3, respectively. Mixtures of SiRFP and SiRHP in a ratio of 8:4 subunits, (lane 4) shift the band to correspond to recombinant SiR (lane 6). Mixing SiRFP and SiRHP in a ratio of 8:8 subunits results in an excess of unbound SiRHP (lane 5). Apo-SiRHP (lane 7) and R83S (lane 8) are significantly larger than WT SiRHP. Neither apo-SiRHP nor R83S binds to SiRFP (lanes 9 and 10). C, log/log plot of hydrodynamic radius of SiR, SiRFP, SiRHP, apo-SiRHP, and R83S determined by DLS versus molecular weight (69) as determined by blue native PAGE.