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. 2020 Aug 11;9:e57081. doi: 10.7554/eLife.57081

Figure 1. Sfh5 is a heme-binding protein.

(A) Cell pellets of E. coli BL21(DE3) culture expressing Sfh5 are reddish brown color (left panel) relative to cell pellets of BL21(DE3) expressing Sec14 (middle panel). Panel at right shows an image of a purified recombinant Sfh5 (500 µM) solution which is distinguished by a deep reddish brown color. (B) UV-vis absorption spectrum of purified Sfh5 is traced in black. Spectra in red identify the spectral profile of Sfh5 after reduction with dithionite. Inset shows the 500 to 650 nm region of the UV-vis spectrum from pyridine hemochromagen assays. The indicated β and α bands at 525 and 557 nm, respectively, diagnose a noncovalently-bound heme b. (C) Top left: UV-vis absorption spectrum of a clarified cell lysate prepared from E. coli expressing Sfh5 absent an N’ terminal 8xHis tag. The spectra were calibrated against a cell lysate prepared in parallel from mock-expressing E. coli cells (vector-only). Top left: The UV-vis absorption spectrum of clarified cell lysate prepared from E. coli BL21 (DE3) cells expressing tag-less Sfh5. Mock lysate prepared from E. coli BL21 (DE3) cells was used as blank to calibrate the spectrophotometer. Both E. coli cultures were induced with 100 µM IPTG and incubated overnight at 16 °C prior to harvesting and lysing cells. Top right: MALDI-TOF mass spectrum signature of hemin (positive standard), Bottom left and bottom right panels show the heme signatures of purified recombinant Sfh5 and Sfh5 purified directly from S. cerevisiae, respectively. Peaks at 616 ± 2 Da constitute a signature for protoporphyrin IX. Total spectral counts within the indicated m/z ranges were as follows: hemin, 30821; recombinant Sfh5, 8038; S. cerevisiae Sfh5, 672. (D, E) UV-vis absorption (left panels) and MALDI-TOF mass spectra (right panels) of purified recombinant Sfh5 sourced from C. albicans (Sfh5CA) and C. glabrata (Sfh5CG). Spectral profiles of dithionite-treated versions of these proteins were red-shifted to 422 and 428 nm for Sfh5CA and Sfh5CG, respectively. Total counts within the indicated m/z ranges were 1404 and 1739 for Sfh5CA and Sfh5CG, respectively.

Figure 1.

Figure 1—figure supplement 1. Sfh5 oligomerization and the unit cell.

Figure 1—figure supplement 1.

Representative analytical ultracentrifugation equilibrium data (gray symbols) and the corresponding global single ideal species fit (solid black lines) are shown for starting concentrations of 10 µM (squares), 5 µM (circles) and 2.5 µM (diamonds) Sfh5 at 15,000 rpm. The residuals of the fit for each concentration are also shown. The estimated molecular mass of the global fit (70,826 Da) corresponds to a dimeric assembly (MWobs/MWcalc = 1.99). When centrifuged at 20,000 RPM, Sfh5 begins to aggregate at the bottom of the cell, and the dimeric single ideal species fit no longer adequately fits the data.