Fig. 5: Heme binding to the ancestral glycosidase is visually apparent.
From: Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase
a The ancestral protein was prepared by Ni-NTA affinity chromatography. The pictures show the samples eluted from the columns for three different preparations that differed by the addition of 20 μM hemin (middle) and 0.4 mM 5-aminolevulinic acid (right) to the culture medium. Neither hemin nor 5-aminolevulinic acid had been added to the culture medium in the preparation on the left. Protein concentrations in these samples were \(\sim\)10 mg/mL. b An ancestral glycosidase sample with a low amount of bound heme (right) was incubated with an excess of heme. A PD10 column and FPLC (fast protein liquid chromatrography) were then used to remove the unbound heme. The resulting protein preparation is shown on the left. Protein concentration is \(\sim\)0.5 mg/mL. c The position of the ancestral protein with bound heme in a FPLC column is revealed by a reddish-brown band.
