The identification of the distinct conformation classes of a molecule is a common and often crucial step in establishing structure-function relationships. Many different methods have been suggested for that purpose which differ in their choice of a (dis)similarity measure and clustering algorithm. The present study discusses and analyzes these issues, proposing a method based on principal component analysis (PCA), which is applied to conformations obtained from molecular dynamics (MD) simulations of an arginylglutamate repeat. Simulations are done at different pH values, using both standard MD and constant-pH MD methods, with the peptide displaying a very high conformational variety. The conformational analysis starts with a comprehensive comparison of different sets of conformational coordinates and of their ability to preserve structural similarity between conformations. The selected set of conformational coordinates is then used to investigate the preservation of structural similarity after PCA transformation, concluding the need of using a multidimensional conformation space. This conformation space is then used to derive a multidimensional probability density and the corresponding energy landscape. The application of a simple cutoff algorithm to the resulting multidimensional landscape is then shown to produce a consistent set of distinct and homogeneous conformation classes. Overall, this methodology provides an efficient way to identify the major conformation classes of a molecule in a way that directly reflects the density of states in the multidimensional conformation space, contrasting with the more heuristic nature of standard clustering methods.

The misfolding of the prion protein (PrP) into a pathogenic β-rich form (PrP(Sc)) has been suggested to occur in the endocytic pathway, triggered by low pH. In this work we performed several constant-pH molecular dynamics simulations of human PrP 90-231 in the pH range 2-7, totaling more than 2 μs. We observed a strong conformational pH dependence where on average the helix content decreased and the β content increased toward acidic pH. Unlike some proposed models, the flexible N-terminus region did not gain stable structure at low pH. Rather, the main structural changes occurred on the helix-rich C-terminus core, as proposed in other models, namely, in the regions around 135-155 and 185-200. The protonation of His187 is found to be associated with a loss of interaction between two PrP subdomains, potentially playing a major role in the misfolding process. In one of the simulations at pH 2, a stable β-rich structure was formed that may be an intermediate of PrP(Sc) formation, indicating that misfolding may precede dimerization.

Under strong sunlight, plants avoid photooxidation by quenching the excess absorbed energy. Quenching is triggered by PsbS, a membrane protein that is activated and deactivated by the light-dependent pH changes in the thylakoid lumen. The mechanism of action of this protein is unknown, but it was suggested that several glutamates act as pH sensors. However, the pKa of glutamate is several pH units below the physiological values in the lumen. Thus, how can PsbS sense the pH of the lumen, and how does it respond to it? By applying a nonstandard molecular dynamics method that treats pH explicitly, we show that the lumen-exposed glutamates of PsbS have strongly shifted pKa values and that such shifts are crucial for the pH sensitivity in physiological conditions. We also demonstrate that protonation drives a systematic unfolding of a region key for protein–protein interactions, indicating that PsbS response to pH is a functional conformational switch.

The pulmonary surfactant protein C (SP-C) is a small highly hydrophobic protein that adopts a mainly helical structure while associated with the membrane but misfolds into a β-rich metastable structure upon deacylation, membrane dissociation, and exposure to the neutral pH of the aqueous alveolar subphase, eventually leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. The present constant-pH MD study of the acylated and deacylated isoforms of SP-C in a chloroform/methanol/water mixture, often used to mimic the membrane environment, shows that the loss of the acyl groups has a structural destabilizing effect and that the increase of pH promotes intraprotein contacts which contribute to the loss of helical structure in solution. These contacts result from the poor solvation of charged groups by the solvent mixture, which exhibits a limited membrane-mimetic character. Although a single SP-C molecule was used in the simulations, we propose that analogous intermolecular interactions may play a role in the early stages of the protein misfolding and aggregation in this mixture.

The prion protein (PrP) is the cause of a group of diseases known as transmissible spongiform encephalopathies (TSEs). Creutzfeldt-Jakob disease and bovine spongiform encephalopathy are examples of TSEs. Although the normal form of PrP (PrP(C)) is monomeric and soluble, it can misfold into a pathogenic form (PrP(Sc)) that has a high content of β-structure and can aggregate forming amyloid fibrils. The mechanism of conversion of PrP(C) into PrP(Sc) is not known but different triggers have been proposed. It can be catalyzed by a PrP(Sc) sample, or it can be induced by an external factor, such as low pH. The pH effect on the structure of PrP was recently studied by computational methods [Campos et al. J. Phys. Chem. B 2010, 114, 12692-12700], and an evident trend of loss of helical structure was observed with pH decrease, together with a gain of β-structures. In particular, one simulation at pH 2 showed an evident misfolding transition. The main goal of the present work was to study the effects of a change in pH to 7 in several transient conformations of this simulation, in order to draw some conclusions about the reversibility of PrP misfolding. Although the most significant effect caused by the change of pH to 7 was a global stabilization of the protein structure, we could also observe that some conformational transitions induced by pH 2 were reversible in many of our simulations, namely those started from the early moments of the misfolding transition. This observation is in good agreement with experiments showing that, even at pH as low as 1.7, it is possible to revert the misfolding process [Bjorndahl et al. Biochemistry 2011, 50, 1162-1173].