The native state simulation of CI2 in pure water at 333 K was stable with a mean Ca RMSD of 2.3 ± 0.4 A over the final 50 ns (Fig. 22.5). The simulation reveals a relatively stable hydrophobic cluster (i.e., all residues in contact 87% of the time) consisting of residues 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. This cluster contains residues from every secondary structure component of CI2 with the exception ofthe active site loop and the turn connecting the second and third b strands (b2 and b3). This cluster is marginally less stable at 333 K than in simulations at lower temperatures (Day et al., 2002). When these residues were not in contact, it was not that water had breached the protein core. Instead dynamic noncore residues had inserted themselves into the core, disrupting packing without unfolding the protein. There is, however, a subset of this cluster that is present 100% of the time at 333 K consisting of residues Ala 16 and Ile 20 in the a helix, Val 47 and Leu 49 in b2, and Pro 61 in b3. These residues describe a concise nucleus expected to be present in native structures of CI2 at 333 K.
In the simulation of CI2 in 7.15 M urea, this reduced hydrophobic core remained intact until 5.429 ns when water began to attack the contact between residues Ile 20 and Val 47 (Fig. 22.5). Before this time, the mean Ca RMSD was 2.5 ± 0.4 A, after which the mean Ca RMSD was 6.3 ± 1.6 A. The large change in Ca RMSD was obvious in the cluster analysis involving multidimensional scaling of the pairwise Ca RMSD matrix (data not shown), which is indicative of an unfolding transition state (TS) (Day and Daggett, 2005; Li and Daggett, 1994, 1996). The mean Ca RMSD of the TS (3.1 A) is somewhat lower than what has been described previously (Day and Daggett, 2005, 2007; Day et al., 2002).
In the starting structure for this simulation, there is one water that is hydrogen bonded to the main chain of residue Leu 49. At 5.429 ns, the TS, water attacks the core and distorts the a helix. Ten picoseconds later (5.439 ns), two more waters have begun to open the gap between the a helix and the b sheet. Additionally, these waters recruit a urea that remains on the periphery of the core until approximately 10.5 ns, when, along with two other urea molecules, it completely disrupts the hydrophobic cluster.
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