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. 2020 Feb 14;3:73. doi: 10.1038/s42003-020-0800-0

Fig. 6. The SPPL2a/b substrates Bri2, FasL, and Tfr1 are heme-binding proteins.

Fig. 6

a Amylose affinity purification of MBP-Bri2-NTF(1–101) and MBP-FasL-TMD(71–113) using Triton X-100 as detergent was followed by SDS-PAGE. b UV/VIS spectra of MBP-Bri2-NTF(1–101) (blue trace) and MBP-FasL-TMD(71–113) (green trace). Addition of imidazole shifts the Soret band of Bri2 from 388 to 418 nm (not shown). c Amylose affinity purification of MBP-LOX-1-NTF(1–88) using DDM as detergent was followed by SDS-PAGE. d MBP-LOX-1-NTF(1–88) (ε280nm = 78840 M−1 cm−1) and MBP-Bri2-NTF(1–101) (ε280nm = 86290 M−1 cm−1) were purified using DDM (not absorbing at 280 nm) as detergent and analyzed before and after concentration by UV/VIS spectroscopy. e Sequence of Tfr1 and SPPL2b cleavage site within the GXXG motif of the TMD (in gray). Residues Cys62 and Cys67 that may contact the heme cofactor are shown in bold red. f Amylose affinity purification of MBP-Tfr1-NTF(1–100) wt and of the C62A and C67A mutants using Triton X-100 as detergent was followed by SDS-PAGE. g UV/VIS spectra of MBP-Tfr1-NTF(1–100) wt (magenta trace, split Soret spectrum with absorbance maxima at 372 and 453 nm) and the mutant proteins C62A (orange trace, Soret band at 384 nm) and C67A (green trace, Soret band at 384 nm). MBP-Tfr1-NTF(1–100) wt purified with DDM as detergent (violet trace) has a single Soret band at 385 nm with a shoulder at about 455 nm (indicated by violet arrow). h Addition of imidazole to purified MBP-Tfr1-NTF(1–100) proteins generates Soret bands at about 360 nm and 420–423 nm, indicating heme coordination by one Cys residue and imidazole. i The heme cofactor of Tfr1 is stably bound as shown by gel filtration using a Superose 6 Increase column [equilibrated in TN buffer containing 0.1% Triton X-100; absorbance is followed at 280 nm (black trace), 370 nm (blue trace), and 453 nm (green trace)]. Results in a–d represent one experiment. f and g show representatives out of two independent experiments, except for purification of MBP-Tfr1-NTF(1–100) using DDM which was done once. h Imidazole titration of wt and mutant MBP-Tfr1-NTF proteins was done once. i Gel filtration of wt protein to show stable heme binding was carried out once.