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Fig. S2. In vitro characterization of ligand binding properties of recombinant ThiY and RibY from C. aurantiacus. (A) Identification of ThiY-bound ligand by MALDI-TOF mass spectrometry. (1) Thiamine (standard) (2) Recombinant ThiY protein with ligand bound (similar signal as free thiamin at m/z 265.207 Da) (3) Recombinant RibY protein (negative control) (4) Buffer alone (TRIS buffer mixed with a-cyano-4-hydroxycinnamic acid matrix) RibY ThiY (1) (2) (3) (4)
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Fig. S2. In vitro characterization of ligand binding properties of recombinant ThiY and RibY from C. aurantiacus. (B) Binding of vitamin ligands tested by fluorescence-based thermal shift (FTS) assay. The positive difference in melting temperatures (ΔT m, °C) between a protein incubated without a ligand and a protein incubated with an increasing ligand concentration was interpreted as potential ligand binding. Points on the plot represent means, and error bars indicate standard deviation values of three independent measurements.
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Fig. S2. In vitro characterization of ligand binding properties of recombinant ThiY and RibY from C. aurantiacus. (C) UV-visible spectra of the recombinant RibY protein and its co-purified ligand. Dark blue, RibY protein with bound co-purified ligand, which is similar to the previously described spectrum of 6,7-dimethyl-8- ribityllumazine (Kaiser et al., 2007). Green, after extraction from the protein by urea denaturation, the RibY ligand showed a similar spectrum. Light blue, free riboflavin (standard). Wavelength (nm) Absorbance
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Fig. S2. In vitro characterization of ligand binding properties of recombinant ThiY and RibY from C. aurantiacus. (D) Identification of RibY-bound ligand by MALDI-TOF mass spectrometry. (1) Buffer alone (TRIS buffer mixed with a-cyano-4-hydroxycinnamic acid) (2) Riboflavin (standard). Three peaks at 377, 378 and 379 Da correspond to the following forms of riboflavin: oxidized and protonated ([376 + 1H] + ), protonated riboflavin radical ([376 + 2H] + ) and protonated reduced riboflavin ([376+3H] + ) (Ohashi, Itoh 2003). (3) Recombinant RibY protein with bound 6,7-dimethyl-8-ribityllumazine. No peaks at the same mass as for riboflavin were detected. RibY (1) (2) (3)
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RibY (1) Riboflavin (standard). No peaks at 326, 327, 328, 329 Da. (2) Recombinant RibY protein with bound 6,7-dimethyl-8-ribityllumazine. Three peaks at 327, 328, and 329 Da correspond to the following forms of 6,7-dimethyl-8-ribityllumazine: oxidized and protonated ([326 + 1H] + ), protonated radical ([326 + 2H] + ), and protonated reduced ([326+3H] + ). The profile has main peak at 328 Da, with the very close to riboflavin distribution of three forms. By analogy with riboflavin, the protonated radical ([326 + 2H] + ) was the most abundant form. (3) Buffer alone (TRIS buffer mixed with a-cyano-4-hydroxycinnamic acid). Fig. S2. In vitro characterization of ligand binding properties of recombinant ThiY and RibY from C. aurantiacus. (D) Identification of RibY-bound ligand by MALDI-TOF mass spectrometry. (1) (2) (3)
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