Protein-bound water calculation 9 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 9
For each box the thermodynamic activity of water was calculated from (eq. 3) and the number of protein-bound water molecules was derived from the size of the largest water cluster (Supporting Table S2). Upon increasing the water activity, water molecules gradually bind to the surface of CALB. The analysis of the MD simulations revealed two distinct phases in the protein hydration: 1) at very low water activity, the water binding sites on the protein surface were occupied by single water molecules ( a ) corresponding to a molar fraction of 54 mol water/mol protein (3% w/w at a w = 0.2). The size of these patches increased with water activity and spanning the hydrophilic surface of the protein, corresponding to a molar fraction of 311 mol water/mol protein (17% w/w at a w = 0.5). However, the hydrophobic substrate binding site was not covered by the spanning water network (b) ; 2) Finally, at high water activity a w > 0.5, the number of protein-bound water molecules rapidly increased, and a continuous multilayer of water molecules on the hydrophilic surface was formed (c) with a thickness of up to 10 Å and a molar fraction of 677 mol water/mol protein (37% w/w at a w = 0.7). Near the active site an isolated water cluster was formed without hydrogen bonding to the spanning water network or the hydrophobic substrate binding site. Simulations with 1708, 2195, and 4091 water molecules showed oversaturation and the formation of pure water droplets. Because the experimental investigations were performed under non-saturating conditions (a w < 1), these simulations were excluded from analysis. In hydration phase I (a w < 0.5) the number of protein-bound waters was proportional to the water activity, since hydration occurs at isolated water binding sites and then gradually a spanning water network was formed which was in agreement with the experimental water sorption isotherms. In hydration phase II (a w > 0.5) the number of protein-bound water molecules became exponential, corresponding to the observed multilayer hydration shell formation. Protein structure and flexibility The root mean square deviation (RMSD) of the CALB backbone atoms from the initial crystallographic structure ranged between 2.2 Å (simulation with 64 water molecules, corresponding to a w = 0.23) and 2.7 Å (simulation with 125 water molecules, corresponding to a w = 0.26). No correlation between a w and RMSD was found, which indicates that there is no systematic conformational change upon increasing the hydration level. Interestingly, the simulation with the highest number of water molecules showed one of the lowest RMSD values (Supporting Figure S3). 10 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 10
Similarly, the global flexibility measured as root mean square fluctuation (RMSF) averaged over all residues seems not to be affected by the hydration level (1.1 - 1.2 Å). However, four regions were identified which were influenced by protein hydration (Supporting Figure S4).
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