The mechanism of denaturation of proteins by urea is explored by using all-atom microseconds molecular dynamics simulations of hen lysozyme generated on BlueGene/L. Accumulation of urea around lysozyme shows that water molecules are expelled from the first hydration shell of the protein. We observe a 2-stage penetration of the protein, with urea penetrating the hydrophobic core before water, forming a ''dry globule.'' The direct dispersion interaction between urea and the protein backbone and side chains is stronger than for water, which gives rise to the intrusion of urea into the protein interior and to urea's preferential binding to all regions of the protein. This is augmented by preferential hydrogen bond formation between the urea carbonyl and the backbone amides that contributes to the breaking of intrabackbone hydrogen bonds. Our study supports the ''direct interaction mechanism'' whereby urea has a stronger dispersion interaction with protein than water.denaturing mechanism ͉ dry globule ͉ molecular dynamics ͉ preferential binding ͉ lysozyme unfolding

SUMMARY The mitochondrial sirtuin SIRT3 regulates metabolic homeostasis during fasting and calorie restriction. We identified mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) as an acetylated protein and a possible target of SIRT3 in a proteomics survey in hepatic mitochondria from Sirt3−/− (SIRT3KO) mice. HMGCS2 is the rate-limiting step in β-hydroxybutyrate synthesis and is hyperacetylated at lysines 310, 447, and 473 in the absence of SIRT3. HMGCS2 is deacetylated by SIRT3 in response to fasting in wild-type mice, but not in SIRT3KO mice. HMGCS2 is deacetylated in vitro when incubated with SIRT3 and in vivo by overexpression of SIRT3. Deacetylation of HMGCS2 lysines 310, 447, and 473 by incubation with wild-type SIRT3 or by mutation to arginine enhances its enzymatic activity. Molecular dynamics simulations show that in silico deacetylation of these three lysines causes conformational changes of HMGCS2 near the active site. Mice lacking SIRT3 show decreased β-hydroxybutyrate levels during fasting. Our findings show SIRT3 regulates ketone body production during fasting and provide molecular insight into how protein acetylation can regulate enzymatic activity.

The role of water in promoting the formation of protofilaments (the basic building blocks of amyloid fibrils) is investigated using fully atomic molecular dynamics simulations. Our model protofilament consists of two parallel β-sheets of Alzheimer Amyloid-β 16-22 peptides (Ac-K 16 -L 17 -V 18 -F 19 -F 20 -A 21 -E 22 -NH 2 ). Each sheet presents a distinct hydrophobic and hydrophilic face and together self-assemble to a stable protofilament with a core consisting of purely hydrophobic residues (L 17 ,F 19 ,A 21 ), with the two charged residues (K 16 , E 22 ) pointing to the solvent. Our simulations reveal a subtle interplay between a water mediated assembly and one driven by favorable energetic interactions between specific residues forming the interior of the protofilament. A dewetting transition, in which water expulsion precedes hydrophobic collapse, is observed for some, but not all molecular dynamics trajectories. In the trajectories in which no dewetting is observed, water expulsion and hydrophobic collapse occur simultaneously, with protofilament assembly driven by direct interactions between the hydrophobic side chains of the peptides (particularly between F-F residues). For those same trajectories, a small increase in the temperature of the simulation (on the order of 20 K) or a modest reduction in the peptide-water van der Waals attraction (on the order of 10%) is sufficient to induce a dewetting transition, suggesting that the existence of a dewetting transition in simulation might be sensitive to the details of the force field parametrization.