It is important to treat electrostatic interactions with accurate methods, such as PME, to avoid potential serious artifacts. It has been shown that choosing simulation parameters, including thermostat and electrostatic treatment, is a subtle issue and that the choice of charge-groups may lead to unphysical effects. Baumketner et al. also reported that charge-group based truncation with reaction-field electrostatics may cause artificial repulsions between charged residues, identified as the microscopic reason behind artificial unfolding of INCB28060 protein in some simulations. Here, charge-groups were chosen to be small to avoid artifacts. Periodic boundary conditions were applied in all directions. To determine whether the binding motifs of ProTa and Neh2 have tendencies to adopt bound-state-like structures in their free states, coordinates from the MD trajectories were compared with the corresponding PDB crystal structures. The Kelch domain of mouse Keap1 was expressed in Escherichia coli BL21 grown in minimal M9 medium. The N-terminally His-tagged protein was purified by affinity chromatography using Ni SepharoseTM 6 Fast Flow beads. The tag was then cleaved by incubation with His-tagged tobacco etch virus protease overnight at 25uC. Cholesterol is an intriguing molecule linked to numerous fundamental physiological functions; thus, alterations in cholesterol metabolism are associated with a range of disorders that include significant current and future healthcare burdens. At least one reason for this extensive linkage of cholesterol to physiological states is that, in contrast to other lipidic membrane components, there are a number of reasonably selective pharmacological tools available with which to modulate the amount and/ or effects of cholesterol in cellular membranes. Indeed, this ability to target native cholesterol with reasonable selectivity, coupled with the availability of sensitive quantitative assays, has led to cholesterol being the first molecule identified as having a direct role in the essential membrane merger steps of fast, calcium-triggered exocytosis, as occurs under physiological conditions, in depolarized nerve terminals. In addition, as a microdomain organizer, cholesterol also plays a critical role in maintaining the localized presence of other components critical to targeting, priming, docking, and subsequent fusion. Among the tools used to study cholesterol, perhaps the most widely applied has been methyl-?-cyclodextrin, a cyclic oligosaccharide that can deplete cellular membranes of cholesterol by increasing the water solubility of the sterol. MbCD has thus been used with increasing frequency to examine the physiological roles of cholesterol. Studies utilizing MbCD have implicated cholesterol in mediating or modulating a wide range of membrane-associated cell BMN673 inquirer properties and functions in a range of secretory cell types, including neurons.