Solute energy based REMD:developments and applications to prion protein misfold predictions

Molecular dynamics (MD) simulations have increasingly contributed to the understanding of biomolecular processes, allowing for predictions of thermodynamic and structural properties. Unfortunately, the holy grail of protein structure prediction was soon found to be severely hampered by the very rugged free energy surface of proteins, with small relative free energies separating native, folded protein conformations from unfolded states. These multiple minima frequently trap present-day protein MD simulations permanently. In order to allow the simulation to escape minima and explore wider portions of conformational space, enhanced sampling techniques were developed. One of the most popular ones, replica exchange molecular dynamics (REMD), is based on multiple parallel MD simulations that are performed with replicas of a system at increasing temperatures T1, T2, etc. Periodic Monte Carlo exchange moves are attempted, aiming to allow conformations to exchange temperature ensembles with a probability that depends on their potential energy and temperature difference. Thus, conformations are simulated at all temperatures and escape local minima with the kinetic energy provided at higher temperatures, while Boltzmann distributions are generated at all temperatures. REMD has been successful in ab-initio folding of a variety of small peptides and proteins (up to 20-30 residues). However, with larger proteins, the overlap of potential energy distributions diminishes, since the potential energy and its fluctuation scale with fkBT, respectively with √fkBT, where f is the number of degrees of freedom of the system, kB Boltzmann's constant and T the temperature. Consequently, the related Monte Carlo exchange probability and number of exchanges in a simulation are also diminished. This is generally compensated by choosing smaller temperature intervals between replicas and thereby increasing the number of necessary replicas (as well as the computational cost of the simulation) to cover a given temperature range. An additional problem relates to explicit solvent simulations, in which solvent to solvent interactions account for the largest part of the total potential energy. Consequently, explicit solvent REMD simulations almost exclusively sample solvent degrees of freedom. These two limitations have lead to the development of REMD protocols for large explicit solvent systems that are based on exchange probabilities computed with subsystem (e.g. protein only) potential energy functions, allowing for a targeted sampling of protein degrees of freedom and a reduction of the computational effort. In this thesis, this approximation is tested by implementing its simplest variation that entirely neglects solvent-solvent interaction as a new REMD protocol termed REM Dpe (Chapter 2). Possible REMD limitations for large explicit solvent systems are tested (Chapter 3) with REM Dpe, which is further applied to perform a thorough and comparative investigation of prion (Chapter 4) and doppel (Chapter 5) protein misfolding. In Chapter 2, the practical validity of the REM Dpe approximation is assessed with simulations of the prion protein, a system that is too large (i.e. 103 residues) to allow for efficient simulations using traditional REMD over the necessary temperature range. A first validation consists in testing whether protein and total potential energy distributions are consistent with their analogs from straightforward reference MD simulations. Second, the overlap pattern of the total potential energy distributions are characterized at different temperatures and show that the exchanges in the REM Dpe simulations are performed according to a Boltzmann weight. Native structures are found to have the lowest protein and total potential energies, as compared to higher energies found for various unfolded structures. Although no obvious bias is detected in the three validations, the conformational landscapes of the REM Dpe simulation at low temperatures progressively shift to non-native regions of the free energy surface. In Chapter 3, this phenomenon is quantified, and its origin identified in insufficient low temperature residence times required for refolding native-like structures. REMD is based on the assumption that systems have to be decorrelated between exchange attempts. Increasing inter-exchange times accordingly would allow for decorrelation and sufficient low temperature residence times but is practically impossible to achieve for large protein simulations, highlighting a major limitation and possible source of bias for present-day REMD simulations. We have chosen the prion as a test case because of its link to transmissible spongiform encephalopathies. Diseases of this category are believed to be caused by a rare prion protein (PrP) misfold leading from the cellular, monomeric, soluble, α-helical PrPC isoform to a pathogenic, aggregated, insoluble, β-rich PrPSc isoform of unknown structure. Gaining experimental knowledge of the PrPSc structure has remained elusive, and aroused interest in predictions supplied by computer simulations. REMD provides a powerful tool allowing to explore a diversity of misfolds and select stable ones that accumulate at lower temperatures. In Chapter 4, we describe a PrP REM Dpe simulation in which rare new β-strands are formed and arrange into a multitude of different β-sheets, reproducing the α-helix → β-sheet conversion observed with circular dichroism spectra. The α-helical and β-sheet propensities along the sequence can thus be computed. We develop and apply the β contact map clustering (bcmc) protocol to identify the most frequent β-sheet pattern defining β-rich folds. 10 new β-rich folds are found and compared to recent experimental data characterizing PrPSc, providing atomistically detailed models for putative monomeric precursors of PrPSc or β-oligomeric conformations. In Chapter 5, an analogous simulation is performed with doppel, a structural homolog of prion (with an identical three α-helix, two β-strand fold) originating from the same gene family, but characterized by a different sequence (only 25% sequence homology), expression pattern and physiological function. Unrelated to amyloid neurodegenerative diseases, doppel supplies the perfect test system to investigate the misfolding of a non-amyloidogenic protein. Prion and doppel misfolding are compared in their monomeric form in the quest to identify prion-specific features that might reveal the mechanism of conversion to PrPSc. In agreement with experiments, we find a lower thermal stability for doppel. Surprisingly, we also observe β-rich forms for doppel. However, the β-rich folds of the two proteins are very different. Moreover, a major difference is found in the free energy barriers leading from the native structure to such conformations as well as to non-native conformations in general: These barriers are low for prion and can already be crossed at 300K, while for doppel they are at least 3 times higher. This difference suggests an intrinsic misfolding and β-enrichment propensity for the monomeric form of prion as compared to doppel

Efficient single-stage optimization of islands in finite-β\beta stellarator equilibria

We present the first single-stage optimization of islands in finite-$\beta$ stellarator equilibria. Stellarator optimization is traditionally performed as a two-stage process; in the first stage, an optimal equilibrium is calculated which balances a set of competing constraints, and in the second stage a set of coils is found that supports said equilibrium. Stage one is generally performed using a representation for the equilibrium that assumes nestedness of flux surfaces, even though this is not warranted and occasionally undesired. The second stage optimization of coils is never perfect, and the mismatch leads to worse performing equilibria, and further deteriorates if additional constraints such as force minimization, coil torsion or port access are included. The higher fidelity of single-stage optimization is especially important for the optimization of islands as these are incredibly sensitive to changes in the field. In this paper we demonstrate an optimization scheme capable of optimizing islands in finite $\beta$ stellarator equilibria directly from coils. We furthermore develop and demonstrate a method to reduce the dimensionality of the single-stage optimization problem to that of the first stage in the two-stage approach.Comment: Submitted to Po

Cycle, productivity and must chemical characteristics of varieties of white wine grape

The objective of this work was to evaluate five varieties of white grapes destined to winemaking, grafted on the two main rootstocks used in viticulture in São Paulo, Brazil, 'IAC 766 Campinas' and 'IAC 572 Jales'. The experiment was performed on the experimental area of Centro APTA de Frutas in the Jundiaí city, São Paulo state, Brazil. The experimental design was a randomized block with split plot with five replications, the plots represented by varieties and subplots by rootstocks. We evaluated the number of days to budding (NDB), number of days to flowering (NDF) and number of days to maturation (NDM), productivity (Prod) (ton.ha−1), content of soluble solids (SS), titratable acidity (TA) and maturation index (MI). There was no difference between rootstocks, and there was interaction among rootstocks and varieties for SS and TA. The varieties did not show significant differences for yield, but there was a significant difference for the other variables. We can concluded that among the assessed varieties on both rootstocks, in Jundiaí / SP, the variety SR 501-17 distinguished itself due to its precocity, productivity and chemical quality of the must

Timing of leaf removal modifies chemical and phenolic composition of Sauvignon Blanc wine

The objective of this work was to evaluate the effect of different defoliation times on the chemical and phenolic composition of Sauvignon Blanc wine in high altitude regions of Santa Catarina - Brazil. The experiment was conducted in the 2015 and 2016 seasons, in a vineyard located in the city of São Joaquim – Santa Catarina State. The defoliation was carried out during phenological stages: full bloom, berries pepper-corn size, pea-sized berries, veraison, 15 days after veraison and control without leaf removal. During harvest, severity of Botrytis cinerea was assessed by visual evaluation through a diagrammatic scale. For the wine samples from each season, total acidity (meq L−1), pH, total polyphenol content (mg L−1 of gallic acid) and color (Abs 420nm) were performed. For the wines of the 2015 harvest, phenolic compounds were analyzed: gallic acid, catechin, vanillic acid, P-coumaric acid and rutin. The chemical composition of Sauvignon Blanc wine was affected as a result of leaf removal timing, defoliation carried out in full bloom, berries pepper-corn size and pea-sized berries reduced total acidity and increased pH of the wines in both seasons. For wine color, in both seasons, it was found a greater yellow coloration in wines originated from grapes where the plants were not defoliated. The increase of yellow coloration is due to the oxidation of catechins and epicatechins, which is related to the increase of botrytis bunch rot. In the 2015 season there were no influence of leaf removal timing in total polyphenols, however, in the 2016, the defoliation performed in full bloom resulted in lower polyphenols contents. Wines from non-defoliated plants presented higher values of catechin, rutin, P-coumaric acid and gallic acid, related to yellow coloration of wines and greater susceptibility to darkening and oxidation. For vanillic acid, wines from non-defoliated plants have the lowest values, this compound is important in wine preservation. Leaf removal is an important management technique in high altitude regions of Santa Catarina - Brazil, it improves wine chemical composition, and if carried out during the stages of full bloom, pepper corn berries and pea-sized berries can reduce the severity of Botrytis bunch rot; and avoid the formation of phenolic compounds related to the processes of oxidation and darkening

Numero di trappole gialle necessarie per stimare nel vigneto la densità relativa delle popolazioni della cicalina Scaphoideus titanus Bali

The density and distribution of immature and adult Scaphoideus titanus Bali on yellow sticky traps was studied in the two vineyards of Castelrotto (Canton of Ticino, Switzerland) and Ghemme (near the town of Novara, Italy). The Aéroxon traps placed horizontally within the plant canopy is most efficient to catch S. titanus adults since they are more mobile then the immature forms.The smallest number of traps assuring a desired reliability of the estimate is calculated on the basis of formai probabilistic statements: half of the lenght of the confidence interval is equal to a proportion D of the standard error of the mean, and the confidence interval should include the true mean with a probability P. Thereby, the variance is expressed as a function of the mean according to Taylor's power law, and the distribution of both the adults and the immature individuals was aggregated. For the purpose of this study, D=0.3 and P=0.9 were assumed to produce reliable estimates. Accordingly, 9 traps are sufficient if the density is 10 specimens per trap, but 41 traps are required fora density of 1 individuals per trap.The two vineyards under study were divided into 6 blocks. Since there were generally no significant differences (P < =O. 05) between these blocks a simple random placement of traps can be envisaged.The method is useful for monitoring S. titanus populations. It permits for example a better estimate of the trials of pesticide applications.La densità e la distribuzione delle forme immature e degli adulti di Scaphoideus titanus sono state studiate in due vigneti: il primo situato nel comune ticinese di Castelrotto, il secondo nel comune novarese di Ghemme. Il metodo di cattura consiste nell'utilizzare le trappole gialle Aéroxon poste orizzontalmente nella vegetazione. Tale sistema è molto piu efficiente nei confronti degli adulti poiché piu mobili delle forme immature.Il minor numero di trappole che assicuri l'attendibilità della stima desiderata, è calcolata sui principi dell'analisi probabilistica formale: metà della lunghezza dell'intervallo di confidenza è uguale al rapporto D dell'errore standard della media e l'intervallo di confidenza deve includere il valore reale della media con una probabilità P. In tal modo la varianza è espressa come funzione della media in accordo con la legge di Taylor. La distribuzione degli adulti e delle forme immature è di tipo aggregato. Per gli scopi di questo studio, D=0.3 e P=0.9 sono stati considerati un buon compromesso per ottenere una stima attendibile. Di conseguenza 9 trappole sono sufficienti se si ha una densità di 10 individui per trappola, ma sono necessarie 41 trappole per una densità di un individuo per trappola.I due vigneti in esame sono stati divisi in 6 blocchi. Generalmente sino a quando non si verificano delle differenze significative (P> = 0.05) tra questi blocchi, si consiglia una semplice randomizzazione delle trappole.Il metodo è utile per il monitoraggio della popolazione di Scaphoideus titanus e permette pure un migliore controllo delle prove di lotta

Comparing floral resource maps and land cover maps to predict predators and aphid suppression on field bean

Context Predatory insects contribute to the natural control of agricultural pests, but also use plant pollen or nectar as supplementary food resources. Resource maps have been proposed as an alternative to land cover maps for prediction of beneficial insects. Objectives We aimed at predicting the abundance of crop pest predating insects and the pest control service they provide with both, detailed flower resource maps and land cover maps. Methods We selected 19 landscapes of 500 m radius and mapped them with both approaches. In the centres of the landscapes, aphid predators – hoverflies (Diptera: Syrphidae), ladybeetles (Coleoptera: Coccinellidae) and lacewings (Neuroptera: Chrysopidae) – were surveyed in experimentally established faba bean phytometers (Vicia faba L. Var. Sutton Dwarf) and their control of introduced black bean aphids (Aphis fabae Scop.) was recorded. Results Landscapes with higher proportions of forest edge as derived from land cover maps supported higher abundance of aphid predators, and high densities of aphid predators reduced aphid infestation on faba bean. Floral resource maps did not significantly predict predator abundance or aphid control services. Conclusions Land cover maps allowed to relate landscape composition with predator abundance, showing positive effects of forest edges. Floral resource maps may have failed to better predict predators because other resources such as overwintering sites or alternative prey potentially play a more important role than floral resources. More research is needed to further improve our understanding of resource requirements beyond floral resource estimations and our understanding of their role for aphid predators at the landscape scale

Comparing floral resource maps and land cover maps to predict predators and aphid suppression on field bean

Context Predatory insects contribute to the natural control of agricultural pests, but also use plant pollen or nectar as supplementary food resources. Resource maps have been proposed as an alternative to land cover maps for prediction of beneficial insects. Objectives We aimed at predicting the abundance of crop pest predating insects and the pest control service they provide with both, detailed flower resource maps and land cover maps. Methods We selected 19 landscapes of 500 m radius and mapped them with both approaches. In the centres of the landscapes, aphid predators – hoverflies (Diptera: Syrphidae), ladybeetles (Coleoptera: Coccinellidae) and lacewings (Neuroptera: Chrysopidae) – were surveyed in experimentally established faba bean phytometers (Vicia faba L. Var. Sutton Dwarf) and their control of introduced black bean aphids (Aphis fabae Scop.) was recorded. Results Landscapes with higher proportions of forest edge as derived from land cover maps supported higher abundance of aphid predators, and high densities of aphid predators reduced aphid infestation on faba bean. Floral resource maps did not significantly predict predator abundance or aphid control services. Conclusions Land cover maps allowed to relate landscape composition with predator abundance, showing positive effects of forest edges. Floral resource maps may have failed to better predict predators because other resources such as overwintering sites or alternative prey potentially play a more important role than floral resources. More research is needed to further improve our understanding of resource requirements beyond floral resource estimations and our understanding of their role for aphid predators at the landscape scale