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Water and urea exchange throughout the rest of the simulation (51 ns) until these core residues are entirely solvated.

The number of residue-to-solvent contacts as a function of simulation time was calculated for all of the specified core residues. The plots for two of these residues, Val 47 and Pro 61, are particularly illustrative of the urea-induced unfolding process (Fig. 22.6). For both residues, water is the first to solvate these residues. In the case of Val 47, the mobilization of water into the core begins almost immediately (Fig. 22.6A). However, for Pro 61 (Fig. 22.6B), hydration occurs shortly (tens of picoseconds) before the unfolding TS at 5.429 ns. After the initial disruption of the core and active site loop, urea molecules also begin to enter the core and eventually replace some water molecules (Fig. 22.6C).

In our simulation of CI2 in 3.87 M TMAO at 333 K, CI2 was stable with a mean Ca RMSD of 2.2 ± 0.6 A over the final 30 ns (Fig. 22.5). The hydrophobic cluster identified from the 333 K pure water simulation of CI2 (Trp 5, Leu 8, Ala 16, Val 19, Ile 20, Ile 29, Val 31, Val 47, Leu 49, Val 51, Ile 57, and Pro 61) was intact 85% of the time. This simulation absolutely conserved the very stable concise hydrophobic nucleus formed by Ala 16, Ile 20, Val 47, Leu 49, and Pro 61. By this metric and others, including the previously mentioned Ca RMSD, CI2 is well folded in 3.87 M TMAO.

The average water residence time in the proximity of polar and charged protein residues was affected by the addition of urea, but not TMAO. CI2 in water and 3.87 M TMAO, waters that were within 3.5 A to polar or charged groups, had an average residence time of 19.2 ± 8.1 and 21.0 ± 6.7 ps. This is in sharp contrast with the lower value from CI2 in 7.15 M urea of 9.6 ± 4.2 ps.

A comparison ofthe percentage ofdouble, single, and no hydrogen donor waters in pure water and CI2 simulations also reveals differences (Table 22.3). CI2, in water alone, has the slight effect of shifting the balance of single and double hydrogen bond donors away from that of pure water toward configurations with less energetically favorable interactions. While this effect is minor with CI2 and water alone (1 to 2% overall), when 7.15 M urea is present the percentage of broken hydrogen bond networks in water increases more dramatically than 8 M urea or CI2 alone. Also of note, the addition of 3.87 M TMAO not only prevents degradation of the hydrogen bonding network by CI2, but yields networks consistent with pure water (sans protein).

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