Protease inhibition by serpins requires a large conformational transition from an active, metastable state to an inactive, stable state. Similar reactions can also occur in the absence of proteases, and these latency transitions take hours, making their time scales many orders of magnitude larger than are currently accessible using conventional molecular dynamics simulations. Using a variational path sampling algorithm, we simulated the entire serpin active-to-latent transition in all-atom detail with a physically realistic force field using a standard computing cluster. These simulations provide a unifying picture explaining existing experimental data for the latency transition of the serpin plasminogen activator inhibitor-1 (PAI-1). They predict a long-lived intermediate that resembles a previously proposed, partially loop-inserted, prelatent state; correctly predict the effects of PAI-1 mutations on the kinetics; and provide a potential means to identify ligands able to accelerate the latency transition. Interestingly, although all of the simulated PAI-1 variants readily access the prelatent intermediate, this conformation is not populated in the active-to-latent transition of another serpin, α 1 -antitrypsin, which does not readily go latent. Thus, these simulations also help elucidate why some inhibitory serpin families are more conformationally labile than others. T he serpin plasminogen activator inhibitor 1 (PAI-1) negatively regulates blood clot clearance (fibrinolysis) by mechanically inhibiting important serine proteases, including tissue type plasminogen activator and urokinase type plasminogen activator (1). Suicide inhibition, initiated by proteolytic cleavage of the PAI-1 reactive center loop (RCL), requires insertion of the cleaved RCL into the central β-sheet (sheet A). This process expands sheet A, inhibits the covalently attached protease by mechanical disruption of the active site (2), and results in a thermodynamically stable serpin conformation. Alternatively, PAI-1 can spontaneously deactivate by inserting its intact, uncleaved RCL into sheet A, resulting in the more stable but inactive latent conformation (1) (Fig. 1).The latency transition provides a facile way to regulate PAI-1 activity. Physiologically, this regulation is achieved by binding to the cell adhesion factor vitronectin, leading to an ∼50% increase in the active state t 1/2 (3). Because high levels of active PAI-1 are associated both with poor prognoses for some cancers, presumably due to interactions with vitronectin, and with cardiovascular diseases (1), PAI-1 inhibitors that accelerate the latency transition are under development (1, 4, 5). However, drug design efforts are hampered by the lack of detailed molecular mechanisms for PAI-1 conformational changes. Numerous studies have identified mutations that either accelerate or retard the conformational transition, as well as antibodies that can accelerate latency. Despite these efforts, the molecular details of the latency transition and the residues involved in the key i...

Ionic surfactants such as sodium dodecyl sulfate (SDS) belong to the amphiphile family: they possess a long hydrophobic hydrocarbon chain and a polar hydrophilic headgroup. In a polar solvent and over the critical micellar concentration these molecules join to form micelles. The micellar solutions, in turn, if doped with various ligands tend to aggregate. Solid SDS, micelles of SDS in water and micelles of SDS doped with two types of macrocyclic ligands, Kryptofix 2.2.2 (K222) and crown ether 18‐Crown‐6 (18C6), at different concentrations are studied by Raman scattering, that represents a new approach to such systems. The experimental Raman spectrum, obtained on crystalline powders of SDS, is compared with the ab initio computed spectrum in order to assign the vibrational bands. After discriminating sensitive peaks by comparing the crystalline powders of the single components and their water solutions, the aggregation process and the action of the ligands are analyzed following the evolution of the intensity and wavenumber of these characteristic Raman peaks. This shows that Raman spectroscopy is sensitive to the aggregation dynamics and to the effects induced by the hydration layer on the molecules in solutions. A saturation effect in the aggregation process with the increase of the ligand concentration is observed. Copyright © 2012 John Wiley & Sons, Ltd.

Im7 and Im9 are evolutionary related proteins with almost identical native structures. In spite of their structural similarity, experiments show that Im7 folds through a long-lived on-pathway intermediate, while Im9 folds according to two-state kinetics. In this work, we use a recently developed enhanced path sampling method to generate many folding trajectories for these proteins, using realistic atomistic force fields, in both implicit and explicit solvent. Overall, our results are in good agreement with the experimental ϕ values and with the result of ϕ-value-restrained molecular dynamics (MD) simulations. However, our implicit solvent simulations fail to predict a qualitative difference in the folding pathways of Im7 and Im9. In contrast, our simulations in explicit solvent correctly reproduce the fact that only protein Im7 folds through a on-pathway intermediate. By analyzing our atomistic trajectories, we provide a physical picture which explains the observed difference in the folding kinetics of these chains.

Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative study of the thermal unfolding of disulfide-rich protein pheromones produced by these ciliates. Recent circular dichroism (CD) measurements have shown that the two psychrophilic (E. nobilii) and mesophilic (E. raikovi) protein families are characterized by very different melting temperatures, despite their close structural homology. The enhanced thermal stability of the E. raikovi pheromones is realized notwithstanding the fact that these proteins form, as a rule, a smaller number of disulfide bonds. We perform Monte Carlo (MC) simulations in a structure-based coarse-grained (CG) model to show that the higher stability of the E. raikovi pheromones is due to the lower locality of the disulfide bonds, which yields a lower entropy increase in the unfolding process. Our study suggests that the higher stability of the mesophilic E. raikovi phermones is not mainly due to the presence of a strongly hydrophobic core, as it was proposed in the literature. In addition, we argue that the molecular adaptation of these ciliates may have occurred from cold to warm, and not from warm to cold. To provide a testable prediction, we identify a point-mutation of an E. nobilii pheromone that should lead to an unfolding temperature typical of that of E. raikovi pheromones.