File S1 - Stability Mechanisms of a Thermophilic Laccase Probed by Molecular Dynamics

Combined supporting information file, containing: Figure S1: RMSD time series for 10 ns NPT equilibration of glycosylated proteins (NAG) in (a) zero ionic strength, (b) 0.3 M NaCl, (c) 0.3 M KF, (d) 1.2 M NaCl, and (e) 1.2 M KF ionic backgrounds. Figure S2: RMSD time series for 10 ns NPT equilibration of proteins without glycosylation (noNAG) in (a) zero ionic strength, (b) 0.3 M NaCl, (c) 0.3 M KF, (d) 1.2 M NaCl, and, (e) 1.2 M KF ionic backgrounds. Figure S3: Backbone RMSD time series for 3 ns NVT simulations in 0.3 M NaCl background extended to 20 ns. (a) 300 K, without glycosylation. (b) 400 K, with glycosylation. (c) 400 K, without glycosylation. The curves include RMSD for the initial 3 ns simulations. Figures S4−S33: Time series of studied properties from 3 ns NVT simulations. a) SASA, b) Backbone RMSD, c) Radius of Gyration, d) Backbone hydrogen bonds. Figures S34−S39: Radial Distribution Functions from the last 3 ns NVT MD for backbone amide-H and halide anions in 0.3 M salt background. Figures S40−S49: B-factor plots for 3 ns NVT MD simulations. Figure S50: Last snapshots from extended (10 ns) NVT simulations at 400 K of TvLαwith and without glycosylation in NaCl backgrounds of 0 M, 0.3 M, and 1.2 M. Figure S51: Backbone hydrogen bond persistence (a) and electrostatic interaction (b) between the involved residues, averaged across the labile hydrogen bond pairs marked in Table S6 and Table S7. Standard deviations are indicated with error-bars. Figure S52: Correlation between backbone hydrogen bond persistence (%) and electrostatic energy (kcal/mol) evaluated between the entire residues for (a) residue pairs in structured parts of the protein (red in Table S8), (b) residue pairs in loosely structured parts of the protein (green in Table S8), and (c) residue pairs in both structured and unstructured parts of the protein. Figure S53: Electrostatic analysis of the C-terminal unfolding observed for glycosylated TvL in zero ionic background at 400 K (b, d, f), but not at 300 K (a, c, e). The time series show electrostatic interactions between three groups: “water” consisting of all TIP3P molecules, “helix” consisting of the last C-terminal residues 489–499, and “protein_nohelix” consisting of the remainder of the protein. Table S1: Statistics calculated from the last 3 ns NVT MD simulations: Average, Standard Deviation, Minimum and Maximum Values for SASA, Rg, and backbone RMSD. Tables S2−S5: Salt bridges found in the last 3 ns NVT MD simulations and their persistence (Cons. %) in percentage of 100 frames sampled from 2 last ns of simulations. Table S6: Loss of persistent hydrogen bonds for glycosylated TvL in 0.3 M NaCl due to the 300 K to 350 K temperature increase. Hydrogen bonds lost in both the 0.3 M and 1.2 M NaCl background are indicated in bold. Table S7: Loss of persistent hydrogen bonds for glycosylated TvL in 1.2 M NaCl due to the 300 K–350 K temperature increase. Hydrogen bonds lost in both the 0.3 M and 1.2 M NaCl background are indicated in bold. Table S8: Simulation averaged electrostatic interaction energy between the residue pairs listed in bold in Table S6 and S7 and persistence of backbone hydrogen bonds for the same residues. The analysis was carried out on the last 2 ns of the 300 K, 350 K, and 400 K simulations of glycosylated TvL in 0.3 M NaCl. Hydrogen bonds in structured and loop regions are indicated in red and green, respectively. Table S9: Persistent backbone hydrogen bonds (HB) calculated for the 500 MD snapshots between t = 10 ns and t = 20 ns in extended NVT simulations.
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