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[Preprint]. 2023 Jun 3:2023.06.02.543507. [Version 1] doi: 10.1101/2023.06.02.543507

Fig. 6. Protonation of the substrate orders the Met20 sidechain in the Michaelis complex.

Fig. 6

50 independent MD simulations of the ecDHFR:NADPH:DHF complex, with and without protonation of the N5 nitrogen, were run for 100 ns each. Kernel density estimates of the (A) hinge distance, (B) Trp22-ϕ, (C) donor-acceptor distance for hydride transfer change upon protonation of the substrate. These kernel density estimates were computed for each trajectory independently and the mean and 95% confidence interval is shown for each condition. (D) The density of water around the N5 nitrogen of DHF as a function of distance from the N5 nitrogen (radial distribution function; RDF) mean and 95% confidence interval are shown. The first 50 frames (20 ns) from one trajectory are superimposed for the (E) deprotonated and (F) protonated substrate, depicting the Met20 sidechain and all waters within 4.5 Å of the N5 nitrogen of DHF. Only the initial frame is depicted for DHF and NADPH for visual clarity.