Fig. 4: Contribution of single/double mutations of mNeonGreen to sulfate sensitivity and MD simulations of the pathways for sulfate entry in Thyone.
a The fluorescence changes of mNeonGreen, mNeonGreen mutants, SulfOFF-1, and Thyone to sulfate upon titration against selected anions at pH 7.4 (n = 6 independent experiments for mNG, mNG-C149I, mNG-S151A, mNG-W167I, mNG-C149I/S151A, Thyone each and n = 3 independent experiments for mNG-C149I/W167I, mNG-S151A/W167I, SulfOFF-1; dots, means; error bars, standard deviations). b Fluorescence changes of mNeonGreen, mNeonGreen mutants, SulfOFF-1, and Thyone to sulfate at 180 mM and pH7.4 (n = 6 independent experiments for mNG, mNG-C149I, mNG-S151A, mNG-W167I, mNG-C149I/S151A, Thyone each and n = 3 independent experiments for mNG-C149I/W167I, mNG-S151A/W167I, SulfOFF each; dots, data points; bars, means; error bars, standard deviations). c The corresponding Kd to sulfate of mNeonGreen, mNeonGreen mutants, SulfOFF-1, and Thyone to sulfate calculated based on the data shown in (a) (n = 6 independent experiments for mNG-C149I, mNG-S151A, mNG-W167I, mNG-C149I/S151A, Thyone each and n = 3 independent experiments for mNG, mNG-C149I/W167I, mNG-S151A/W167I, SulfOFF-1 each; dots, data points; bars, means; error bars, standard deviations). d The predicted entry pathways of Thyone through constant pH MD simulations. Pathway 1, outlined in orange: Sulfate initially binds to surface residues K166, K152, and R150. When the sulfate is moved between the proximal β sheets, after 20 ns the sulfate is bound inside close to the original pocket. Pathway 2, outlined in blue: Sulfate initially binds between β sheets 7 and 10 to K152, K153 and R150. When the sulfate is moved between the proximal beta sheets, after 20 ns the sulfate was bound with K153, V203 and H72 partially inside the β barrel. The final bound position of sulfate is shown in the central panel. Hydrogen bonds are shown in cyan dashed lines, with residues shown in gray stick, sulfate shown in colored stick, and the chromophore shown in yellow stick. e Structural changes upon binding of sulfate to Thyone and W167I systems, with the original position of the chromophore shown in yellow stick, its position with sulfate bound shown in gray stick, binding residues shown in green stick, sulfate residue shown in colored stick, and hydrogen bonds shown in dashed cyan. (1) Representative structure of Thyone in transparent green cartoon, with mutated positions are highlighted in pink (I167) and purple (S151 and C149). (2) Zoom of labeled binding pocket of Thyone. (3) The change in chromophore position upon binding of sulfate to Thyone. (4) The beta barrel structure of W167I where mutated position is highlighted in pink (I167). (5) Zoom of labeled binding pocket of W167I. (6) The change in chromophore position upon binding of sulfate to W167I.
