Search

Plants in habitats with unpredictable conditions often have diversified bet-hedging strategies that ensure fitness over a wider range of variable environmental factors. A striking example is the diaspore (seed and fruit) heteromorphism that evolved to maximize species survival in Aethionema arabicum (Brassicaceae) in which external and endogenous triggers allow the production of two distinct diaspores on the same plant. Using this dimorphic diaspore model, we identified contrasting molecular, biophysical, and ecophysiological mechanisms in the germination responses to different temperatures of the mucilaginous seeds (M+ seed morphs), the dispersed indehiscent fruits (IND fruit morphs), and the bare non-mucilaginous M- seeds obtained by pericarp (fruit coat) removal from IND fruits. Large-scale comparative transcriptome and hormone analyses of M+ seeds, IND fruits, and M- seeds provided comprehensive datasets for their distinct thermal responses. Morph-specific differences in co-expressed gene modules in seeds, as well as in seed and pericarp hormone contents, identified a role of the IND pericarp in imposing coat dormancy by generating hypoxia affecting abscisic acid (ABA) sensitivity. This involved expression of morph-specific transcription factors, hypoxia response, and cell wall remodeling genes, as well as altered ABA metabolism, transport, and signaling. Parental temperature affected ABA contents and ABA-related gene expression and altered IND pericarp biomechanical properties. Elucidating the molecular framework underlying the diaspore heteromorphism can provide insight into developmental responses to globally changing temperatures., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Background: The evolutionary success of flowering plants is associated with the vast diversity of their reproductive structures. Despite recent progress in understanding angiosperm-wide trends of floral structure and evolution, a synthetic view of the diversity in seed form and function across angiosperms is lacking., Scope: Here we present a roadmap to synthesise the diversity of seed forms in extant angiosperms, relying on the morphospace concept, i.e. a mathematical representation which relates multiple traits and describes the realised morphologies. We provide recommendations on how to broaden the range of measurable traits beyond mass, by using key morphological traits representative of the embryo, endosperm, and seed coat but also fruit attributes (e.g., dehiscence, fleshiness). These key traits were used to construct and analyse a morphospace to detect evolutionary trends and gain insight into how morphological traits relate to seed functions. Finally, we outline challenges and future research directions, combining the morphospace with macroevolutionary comparative methods to underline the drivers that gave rise to the diversity of observed seed forms., Conclusions: We conclude that this multidimensional approach has the potential, although still untapped, to improve our understanding of covariation among reproductive traits, and further elucidate angiosperm reproductive biology as a whole., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The transition from germinating seeds to emerging seedlings is one of the most vulnerable plant life cycle stages. Heteromorphic diaspores (seed and fruit dispersal units) are an adaptive bet-hedging strategy to cope with spatiotemporally variable environments. While the roles and mechanisms of seedling traits have been studied in monomorphic species, which produce one type of diaspore, very little is known about seedlings in heteromorphic species. Using the dimorphic diaspore model Aethionema arabicum (Brassicaceae), we identified contrasting mechanisms in the germination responses to different temperatures of the mucilaginous seeds (M + seed morphs), the dispersed indehiscent fruits (IND fruit morphs), and the bare non-mucilaginous M - seeds obtained from IND fruits by pericarp (fruit coat) removal. What follows the completion of germination is the pre-emergence seedling growth phase, which we investigated by comparative growth assays of early seedlings derived from the M + seeds, bare M - seeds, and IND fruits. The dimorphic seedlings derived from M + and M - seeds did not differ in their responses to ambient temperature and water potential. The phenotype of seedlings derived from IND fruits differed in that they had bent hypocotyls and their shoot and root growth was slower, but the biomechanical hypocotyl properties of 15-day-old seedlings did not differ between seedlings derived from germinated M + seeds, M - seeds, or IND fruits. Comparison of the transcriptomes of the natural dimorphic diaspores, M + seeds and IND fruits, identified 2,682 differentially expressed genes (DEGs) during late germination. During the subsequent 3 days of seedling pre-emergence growth, the number of DEGs was reduced 10-fold to 277 root DEGs and 16-fold to 164 shoot DEGs. Among the DEGs in early seedlings were hormonal regulators, in particular for auxin, ethylene, and gibberellins. Furthermore, DEGs were identified for water and ion transporters, nitrate transporter and assimilation enzymes, and cell wall remodeling protein genes encoding enzymes targeting xyloglucan and pectin. We conclude that the transcriptomes of seedlings derived from the dimorphic diaspores, M + seeds and IND fruits, undergo transcriptional resetting during the post-germination pre-emergence growth transition phase from germinated diaspores to growing seedlings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Arshad, Steinbrecher, Wilhelmsson, Fernandez-Pozo, Pérez, Mérai, Rensing, Chandler and Leubner-Metzger.)

Main Conclusion: Seed priming with gas plasma-activated water results in an increased ageing resilience in Eragrostis tef grains compared to a conventional hydropriming protocol. Tef (Eragrostis tef) is a cereal grass and a major staple crop of Ethiopia and Eritrea. Despite its significant importance in terms of production, consumption, and cash crop value, tef has been understudied and its productivity is low. In this study, tef grains have undergone different priming treatments to enhance seed vigour and seedling performance. A conventional hydropriming and a novel additive priming technology with gas plasma-activated water (GPAW) have been used and tef grains were then subjected to germination performance assays and accelerated ageing. Tef priming increases the germination speed and vigour of the grains. Priming with GPAW retained the seed storage potential after ageing, therefore, presenting an innovative environmental-friendly seed technology with the prospect to address variable weather conditions and ultimately food insecurity. Seed technology opens new possibilities to increase productivity of tef crop farming to achieve a secure and resilient tef food system and economic growth in Ethiopia by sustainable intensification of agriculture beyond breeding., (© 2024. The Author(s).)

Developing innovative agri-technologies is essential for the sustainable intensification of global food production. Seed dormancy is an adaptive trait which defines the environmental conditions in which the seed is able to germinate. Dormancy release requires sensing and integration of multiple environmental signals, a complex process which may be mimicked by seed treatment technologies. Here, we reveal molecular mechanisms by which non-thermal (cold) atmospheric gas plasma-activated water (GPAW) releases the physiological seed dormancy of Arabidopsis thaliana. GPAW triggered dormancy release by synergistic interaction between plasma-generated reactive chemical species (NO3-, H2O2, ·NO, and ·OH) and multiple signalling pathways targeting gibberellin and abscisic acid (ABA) metabolism and the expression of downstream cell wall-remodelling genes. Direct chemical action of GPAW on cell walls resulted in premature biomechanical endosperm weakening. The germination responses of dormancy signalling (nlp8, prt6, and dog1) and ABA metabolism (cyp707a2) mutants varied with GPAW composition. GPAW removes seed dormancy blocks by triggering multiple molecular signalling pathways combined with direct chemical tissue weakening to permit seed germination. Gas plasma technologies therefore improve seed quality by mimicking permissive environments in which sensing and integration of multiple signals lead to dormancy release and germination., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Seeds sense temperature, nutrient levels and light conditions to inform decision making on the timing of germination. Limited light availability for photoblastic species results in irregular germination timing and losses of population germination percentage. Seed industries are therefore looking for interventions to mitigate this risk. A growing area of research is water treated with gas plasma (GPAW), in which the formed solution is a complex consisting of reactive oxygen and nitrogen species. Gas plasma technology is widely used for sterilisation and is an emerging technology in the food processing industry. The use of the GPAW on seeds has previously led to an increase in germination performance, often attributed to bolstered antioxidant defence mechanisms. However, there is a limited understanding of how the solution may influence the mechanisms that govern seed dormancy and whether photoreceptor-driven germination mechanisms are affected. In our work, we studied how GPAW can influence the mechanisms that govern photo-dependent dormancy, isolating the effects at low fluence response (LFR) and very low fluence response (VLFR). The two defined light intensity thresholds affect germination through different phytochrome photoreceptors, PHYB and PHYA, respectively; we found that GPAW showed a significant increase in population germination percentage under VLFR and further described how each treatment affects key physiological regulators.

The dynamic behaviour of seeds in soil seed banks depends on their ability to act as sophisticated environmental sensors to adjust their sensitivity thresholds for germination by dormancy mechanisms. Here we show that prolonged incubation of sugar beet fruits at low temperature (chilling at 5°C, generally known to release seed dormancy of many species) can induce secondary nondeep physiological dormancy of an apparently nondormant crop species. The physiological and biophysical mechanisms underpinning this cold-induced secondary dormancy include the chilling-induced accumulation of abscisic acid in the seeds, a reduction in the embryo growth potential and a block in weakening of the endosperm covering the embryonic root. Transcriptome analysis revealed distinct gene expression patterns in the different temperature regimes and upon secondary dormancy induction and maintenance. The chilling caused reduced expression of cell wall remodelling protein genes required for embryo cell elongation growth and endosperm weakening, as well as increased expression of seed maturation genes, such as for late embryogenesis abundant proteins. A model integrating the hormonal signalling and master regulator expression with the temperature-control of seed dormancy and maturation programmes is proposed. The revealed mechanisms of the cold-induced secondary dormancy are important for climate-smart agriculture and food security., (© 2022 The Authors, Plant, Cell & Environment published by Wiley & Sons Ltd.)

Underdeveloped (small) embryos embedded in abundant endosperm tissue, and thus having morphological dormancy (MD) or morphophysiological dormancy (MPD), are considered to be the ancestral state in seed dormancy evolution. This trait is retained in the Apiaceae family, which provides excellent model systems for investigating the underpinning mechanisms. We investigated Apium graveolens (celery) MD by combined innovative imaging and embryo growth assays with the quantification of hormone metabolism, as well as the analysis of hormone and cell-wall related gene expression. The integrated experimental results demonstrated that embryo growth occurred inside imbibed celery fruits in association with endosperm degradation, and that a critical embryo size was required for radicle emergence. The regulation of these processes depends on gene expression leading to gibberellin and indole-3-acetic acid (IAA) production by the embryo and on crosstalk between the fruit compartments. ABA degradation associated with distinct spatiotemporal patterns in ABA sensitivity control embryo growth, endosperm breakdown and radicle emergence. This complex interaction between gibberellins, IAA and ABA metabolism, and changes in the tissue-specific sensitivities to these hormones is distinct from non-MD seeds. We conclude that the embryo growth to reach the critical size and the associated endosperm breakdown inside MD fruits constitute a unique germination programme., (© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

In this study, we generated a matched molecular pair dataset of halogen/deshalogen compounds with reliable binding affinity data and structural binding mode information from public databases. The workflow includes automated system preparation and setup of free energy perturbation relative binding free energy calculations. We demonstrate the suitability of these datasets to investigate the performance of molecular mechanics force fields and molecular simulation algorithms for the purpose of in silico affinity predictions in lead optimization. Our datasets of a total of 115 matched molecular pairs show highly accurate binding free energy predictions with an average error of <1 kcal/mol despite the semi-automated calculation scheme. We quantify the accuracy of the optimized potential for liquid simulations (OPLS) force field to predict the effect of halogen addition to compounds, a commonly employed chemical modification in the design of drug-like molecules.

The developmental transition from a fertilized ovule to a dispersed diaspore (seed or fruit) involves complex differentiation processes of the ovule's integuments leading to the diversity in mature seed coat structures in angiosperms. In this study, comparative imaging and transcriptome analysis were combined to investigate the morph-specific developmental differences during outer seed coat differentiation and mucilage production in Aethionema arabicum, the Brassicaceae model for diaspore dimorphism. One of the intriguing adaptations of this species is the production and dispersal of morphologically distinct, mucilaginous and non-mucilaginous diaspores from the same plant (dimorphism). The dehiscent fruit morph programme producing multiple mucilaginous seed diaspores was used as the default trait combination, similar to Arabidopsis thaliana, and was compared with the indehiscent fruit morph programme leading to non-mucilaginous diaspores. Synchrotron-based radiation X-ray tomographic microscopy revealed a co-ordinated framework of morph-specific early changes in internal anatomy of developing A. arabicum gynoecia including seed abortion in the indehiscent programme and mucilage production by the mucilaginous seed coat. The associated comparative analysis of the gene expression patterns revealed that the unique seed coat dimorphism of Ae. arabicum provides an excellent model system for comparative study of the control of epidermal cell differentiation and mucilage biosynthesis by the mucilage transcription factor cascade and their downstream cell wall and mucilage remodelling genes. Elucidating the underlying molecular framework of the dimorphic diaspore syndrome is key to understanding differential regulation of bet-hedging survival strategies in challenging environments, timely in the face of global climatic change., (© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

How the biophysical properties of overlaying tissues control growth, such as the embryonic root (radicle) during seed germination, is a fundamental question. In eudicot seeds the endosperm surrounding the radicle confers coat dormancy and controls germination responses through modulation of its cell wall mechanical properties. Far less is known for grass caryopses that differ in tissue morphology. Here we report that the coleorhiza, a sheath-like organ that surrounds the radicle in grass embryos, performs the same role in the grass weed Avena fatua (common wild oat). We combined innovative biomechanical techniques, tissue ablation, microscopy, tissue-specific gene and enzyme activity expression with the analysis of hormones and oligosaccharides. The combined experimental work demonstrates that in grass caryopses the coleorhiza indeed controls germination for which we provide direct biomechanical evidence. We show that the coleorhiza becomes reinforced during dormancy maintenance and weakened during germination. Xyloglucan endotransglycosylases/hydrolases may have a role in coleorhiza reinforcement through cell wall remodelling to confer coat dormancy. The control of germination by coleorhiza-enforced dormancy in grasses is an example of the convergent evolution of mechanical restraint by overlaying tissues., (©2020 The Authors. New Phytologist ©2020 New Phytologist Trust.)

Accurate ranking of compounds with regards to their binding affinity to a protein using computational methods is of great interest to pharmaceutical research. Physics-based free energy calculations are regarded as the most rigorous way to estimate binding affinity. In recent years, many retrospective studies carried out both in academia and industry have demonstrated its potential. Here, we present the results of large-scale prospective application of the FEP+ method in active drug discovery projects in an industry setting at Merck KGaA, Darmstadt, Germany. We compare these prospective data to results obtained on a new diverse, public benchmark of eight pharmaceutically relevant targets. Our results offer insights into the challenges faced when using free energy calculations in real-life drug discovery projects and identify limitations that could be tackled by future method development. The new public data set we provide to the community can support further method development and comparative benchmarking of free energy calculations.

Hypophosphatasia (HPP) is a rare metabolic disorder characterized by low tissue-nonspecific alkaline phosphatase (TNSALP) typically caused by ALPL gene mutations. HPP is heterogeneous, with clinical presentation correlating with residual TNSALP activity and/or dominant-negative effects (DNE). We measured residual activity and DNE for 155 ALPL variants by transient transfection and TNSALP enzymatic activity measurement. Ninety variants showed low residual activity and 24 showed DNE. These results encompass all missense variants with carrier frequencies above 1/25,000 from the Genome Aggregation Database. We used resulting data as a reference to develop a new computational algorithm that scores ALPL missense variants and predicts high/low TNSALP enzymatic activity. Our approach measures the effects of amino acid changes on TNSALP dimer stability with a physics-based implicit solvent energy model. We predict mutation deleteriousness with high specificity, achieving a true-positive rate of 0.63 with false-positive rate of 0, with an area under receiver operating curve (AUC) of 0.9, better than all in silico predictors tested. Combining this algorithm with other in silico approaches can further increase performance, reaching an AUC of 0.94. This study expands our understanding of HPP heterogeneity and genotype/phenotype relationships with the aim of improving clinical ALPL variant interpretation., (© 2020 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

Main Conclusion: Storage at an elevated partial pressure of oxygen and classical artificial ageing cause a rapid loss of seed viability of short-lived vegetable seeds. Prolonging seed longevity during storage is of major importance for gene banks and the horticultural industry. Slowing down biochemical deterioration, including oxygen-dependent deterioration caused by oxidative processes can boost longevity. This can be affected by the seed structure and the oxygen permeability of seed coat layers. Classical artificial seed ageing assays are used to estimate seed 'shelf-life' by mimicking seed ageing via incubating seeds at elevated temperature and elevated relative humidity (causing elevated equilibrium seed moisture content). In this study, we show that seed lots of vegetable Allium species are short-lived both during dry storage for several months and in seed ageing assays at elevated seed moisture levels. Micromorphological analysis of the Allium cepa x Allium fistulosum salad onion seed identified intact seed coat and endosperm layers. Allium seeds equilibrated at 70% relative humidity were used to investigate seed ageing at tenfold elevated partial pressure of oxygen (high pO 2 ) at room temperature (22 ºC) in comparison to classical artificial ageing at elevated temperature (42 ºC). Our results reveal that 30 days high pO 2 treatment causes a rapid loss of seed viability which quantitatively corresponded to the seed viability loss observed by ~ 7 days classical artificial ageing. A similar number of normal seedlings develop from the germinating (viable) proportion of seeds in the population. Many long-lived seeds first exhibit a seed vigour loss, evident from a reduced germination speed, preceding the loss in seed viability. In contrast to this, seed ageing of our short-lived Allium vegetable seems to be characterised by a rapid loss in seed viability.

Main Conclusion: Seed-processing technologies such as polishing and washing enhance crop seed quality by limited removal of the outer layers and by leaching. Combined, this removes chemical compounds that inhibit germination. Industrial processing to deliver high-quality commercial seed includes removing chemical inhibitors of germination, and is essential to produce fresh sprouts, achieve vigorous crop establishment, and high yield potential in the field. Sugar beet (Beta vulgaris subsp. vulgaris var. altissima Doell.), the main sugar source of the temperate agricultural zone, routinely undergoes several processing steps during seed production to improve germination performance and seedling growth. Germination assays and seedling phenotyping was carried out on unprocessed, and processed (polished and washed) sugar beet fruits. Pericarp-derived solutes, known to inhibit germination, were tested in germination assays and their osmolality and conductivity assessed (ions). Abscisic acid (ABA) and ABA metabolites were quantified in both the true seed and pericarp tissue using UPLC-ESI(+)-MS/MS. Physical changes in the pericarp structures were assessed using scanning electron microscopy (SEM). We found that polishing and washing of the sugar beet fruits both had a positive effect on germination performance and seedling phenotype, and when combined, this positive effect was stronger. The mechanical action of polishing removed the outer pericarp (fruit coat) tissue (parenchyma), leaving the inner tissue (sclerenchyma) unaltered, as revealed by SEM. Polishing as well as washing removed germination inhibitors from the pericarp, specifically, ABA, ABA metabolites, and ions. Understanding the biochemistry underpinning the effectiveness of these processing treatments is key to driving further innovations in commercial seed quality.

Heteromorphic diaspores (fruits and seeds) are an adaptive bet-hedging strategy to cope with spatiotemporally variable environments, particularly fluctuations in favourable temperatures and unpredictable precipitation regimes in arid climates. We conducted comparative analyses of the biophysical and ecophysiological properties of the two distinct diaspores (mucilaginous seed (M + ) vs indehiscent (IND) fruit) in the dimorphic annual Aethionema arabicum (Brassicaceae), linking fruit biomechanics, dispersal aerodynamics, pericarp-imposed dormancy, diaspore abscisic acid (ABA) concentration, and phenotypic plasticity of dimorphic diaspore production to its natural habitat and climate. Two very contrasting dispersal mechanisms of the A. arabicum dimorphic diaspores were revealed. Dehiscence of large fruits leads to the release of M + seed diaspores, which adhere to substrata via seed coat mucilage, thereby preventing dispersal (antitelechory). IND fruit diaspores (containing nonmucilaginous seeds) disperse by wind or water currents, promoting dispersal (telechory) over a longer range. The pericarp properties confer enhanced dispersal ability and degree of dormancy on the IND fruit morph to support telechory, while the M + seed morph supports antitelechory. Combined with the phenotypic plasticity to produce more IND fruit diaspores in colder temperatures, this constitutes a bet-hedging survival strategy to magnify the prevalence in response to selection pressures acting over hilly terrain., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

We present a molecular dynamics simulation study of alkali metal cation transport through the double-helical and the head-to-head conformers of the gramicidin ion channel. Our approach is based on a thermodynamic integration network, which consists of a sequence of transport reactions, absolute free energies of solvation and cycles of alchemical transmutations of the ions. In this manner, we can reliably estimate free energies and their statistical errors via a least-squares method without imposing external forces on the system. Within the double helical channel, we find a free energy surface typical for hopping transport between isoenergetic sites of ion localization, separated by comparatively large activation barriers. For fast transport through the head-to-head conformation, the thermodynamic network scheme starts to break down. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

The membrane alignment of helical amphiphilic peptides in oriented phospholipid bilayers can be obtained as ensemble and time averages from solid state 2 H NMR by fitting the quadrupolar splittings to ideal α-helices. At the same time, molecular dynamics (MD) simulations can provide atomistic insight into peptide-membrane systems. Here, we evaluate the potential of MD simulations to complement the experimental NMR data that is available on three exemplary systems: the natural antimicrobial peptide PGLa and the two designer-made peptides MSI-103 and KIA14, whose sequences were derived from PGLa. Each peptide was simulated for 1 μs in a DMPC lipid bilayer. We calculated from the MD simulations the local angles which define the side chain geometry with respect to the peptide helix. The peptide orientation was then calculated (i) directly from the simulation, (ii) from back-calculated MD-derived NMR splittings, and (iii) from experimental 2 H NMR splittings. Our findings are that (1) the membrane orientation and secondary structure of the peptides found in the NMR analysis are generally well reproduced by the simulations; (2) the geometry of the side chains with respect to the helix backbone can deviate significantly from the ideal structure depending on the specific residue, but on average all side chains have the same orientation; and (3) for all of our peptides, the azimuthal rotation angle found from the MD-derived splittings is about 15° smaller than the experimental value.

Distinct plant seed/fruit structures evolved to support reproduction and dispersal in distinct environments. Appropriate biomechanical properties and interactions of the various seed compartments are indispensable to plant survival. Most seeds are dispersed in a dry state generated during seed development/maturation for which novel aspects of endosperm-embryo interaction were discovered. The various layers covering the embryo of a mature seed define the patterns of water uptake during germination. Their biomechanical weakening together with embryo cell expansion is mediated by cell wall remodelling to facilitate radicle protrusion. Recent work with different species has revealed mechanisms underpinning specific embryo growth zones. Abiotic and biotic factors were shown to release different types of seed and fruit coat-mediated constraints to water uptake and germination., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Estimating the correct binding modes of ligands in protein-ligand complexes is crucial not only in the drug discovery process but also for elucidating potential toxicity mechanisms. In the current paper, we propose a computational modeling workflow using the combination of docking, classical molecular dynamics (cMD), accelerated molecular dynamics (aMD) and free-energy perturbation (FEP+ protocol) for identification of possible ligand binding modes. It was applied for investigation of selected perfluorocarboxyl acids (PFCAs) in the PPARγ nuclear receptor. Although both regular and induced fit docking failed to reproduce the experimentally determined binding mode of the ligands when docked into a non-native X-ray structure, cMD and aMD simulations successfully identified the most probable binding conformations. Moreover, multiple binding modes were identified for all of these compounds and the shorter-chain PFCAs continuously moved between a few energetically favorable binding conformations. On the basis of MD predictions of binding conformations, we applied the default and also redesigned FEP+ sampling protocols, which accurately reproduced experimental differences in the binding energies. Thus, the preliminary MD simulations can also provide helpful information about correct setup of the FEP+ calculations. These results show that the PFCA binding modes were accurately predicted and that the FEP+ protocol can be used to estimate free energies of binding of flexible ligands that are not typical druglike compounds. Our in silico workflow revealed the specific ligand-residue interactions within the ligand binding domain and the main characteristics of the PFCAs, and it was concluded that these compounds are week PPARγ partial agonists. This work also suggests a common pipeline for identification of ligand binding modes, ligand-protein dynamics description, and relative free-energy calculations., Competing Interests: The authors declare no competing financial interest.

Optimization of fragment size D-amino acid oxidase (DAAO) inhibitors was investigated using a combination of computational and experimental methods. Retrospective free energy perturbation (FEP) calculations were performed for benzo[d]isoxazole derivatives, a series of known inhibitors with two potential binding modes derived from X-ray structures of other DAAO inhibitors. The good agreement between experimental and computed binding free energies in only one of the hypothesized binding modes strongly support this bioactive conformation. Then, a series of 1-H-indazol-3-ol derivatives formerly not described as DAAO inhibitors was investigated. Binding geometries could be reliably identified by structural similarity to benzo[d]isoxazole and other well characterized series and FEP calculations were performed for several tautomers of the deprotonated and protonated compounds since all these forms are potentially present owing to the experimental pKa values of representative compounds in the series. Deprotonated compounds are proposed to be the most important bound species owing to the significantly better agreement between their calculated and measured affinities compared to the protonated forms. FEP calculations were also used for the prediction of the affinities of compounds not previously tested as DAAO inhibitors and for a comparative structure-activity relationship study of the benzo[d]isoxazole and indazole series. Selected indazole derivatives were synthesized and their measured binding affinity towards DAAO was in good agreement with FEP predictions.

The therapeutic effect of targeted kinase inhibitors can be significantly reduced by intrinsic or acquired resistance mutations that modulate the affinity of the drug for the kinase. In cancer, the majority of missense mutations are rare, making it difficult to predict their impact on inhibitor affinity. This complicates the practice of precision medicine, pairing of patients with clinical trials, and development of next-generation inhibitors. Here, we examine the potential for alchemical free-energy calculations to predict how kinase mutations modulate inhibitor affinities to Abl, a major target in chronic myelogenous leukemia (CML). We find these calculations can achieve useful accuracy in predicting resistance for a set of eight FDA-approved kinase inhibitors across 144 clinically-identified point mutations, achieving a root mean square error in binding free energy changes of 1.1 0.9 1.3 kcal/mol (95% confidence interval) and correctly classifying mutations as resistant or susceptible with 88 82 93 % accuracy. Since these calculations are fast on modern GPUs, this benchmark establishes the potential for physical modeling to collaboratively support the rapid assessment and anticipation of the potential for patient mutations to affect drug potency in clinical applications.

The biomechanical and ecophysiological properties of plant seed/fruit structures are fundamental to survival in distinct environments. Dispersal of fruits with hard pericarps (fruit coats) encasing seeds has evolved many times independently within taxa that have seed dispersal as their default strategy. The mechanisms by which the constraint of a hard pericarp determines germination timing in response to the environment are currently unknown. Here, we show that the hard pericarp of Lepidium didymum controls germination solely by a biomechanical mechanism. Mechanical dormancy is conferred by preventing full phase-II water uptake of the encased non-dormant seed. The lignified endocarp has biomechanically and morphologically distinct regions that serve as predetermined breaking zones. This pericarp-imposed mechanical dormancy is released by the activity of common fungi, which weaken these zones by degrading non-lignified pericarp cells. We propose that the hard pericarp with this biomechanical mechanism contributed to the global distribution of this species in distinct environments.

Protein side-chain mutation is fundamental both to natural evolutionary processes and to the engineering of protein therapeutics, which constitute an increasing fraction of important medications. Molecular simulation enables the prediction of the effects of mutation on properties such as binding affinity, secondary and tertiary structure, conformational dynamics, and thermal stability. A number of widely differing approaches have been applied to these predictions, including sequence-based algorithms, knowledge-based potential functions, and all-atom molecular mechanics calculations. Free energy perturbation theory, employing all-atom and explicit-solvent molecular dynamics simulations, is a rigorous physics-based approach for calculating thermodynamic effects of, for example, protein side-chain mutations. Over the past several years, we have initiated an investigation of the ability of our most recent free energy perturbation methodology to model the thermodynamics of protein mutation for two specific problems: protein-protein binding affinities and protein thermal stability. We highlight recent advances in the field and outline current and future challenges., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The stability of folded proteins is critical to their biological function and for the efficacy of protein therapeutics. Predicting the energetic effects of protein mutations can improve our fundamental understanding of structural biology, the molecular basis of diseases, and possible routes to addressing those diseases with biological drugs. Identifying the effect of single amino acid point mutations on the thermodynamic equilibrium between the folded and unfolded states of a protein can pinpoint residues of critical importance that should be avoided in the process of improving other properties (affinity, solubility, viscosity, etc.) and suggest changes at other positions for increasing stability in protein engineering. Multiple computational tools have been developed for in silico predictions of protein stability in recent years, ranging from sequence-based empirical approaches to rigorous physics-based free energy methods. In this work, we show that FEP+, which is a free energy perturbation method based on all-atom molecular dynamics simulations, can provide accurate thermal stability predictions for a wide range of biologically relevant systems. Significantly, the FEP+ approach, while originally developed for relative binding free energies of small molecules to proteins and not specifically fitted for protein stability calculations, performs well compared to other methods that were fitted specifically to predict protein stability. Here, we present the broadest validation of a rigorous free energy-based approach applied to protein stability reported to date: 700+ single-point mutations spanning 10 different protein targets. Across the entire data set, we correctly classify the mutations as stabilizing or destabilizing in 84% of the cases, and obtain statistically significant predictions as compared with experiment [average error of ~1.6kcal/mol and coefficient of determination (R 2 ) of 0.40]. This study demonstrates, for the first time in a large-scale validation, that rigorous free energy calculations can be used to predict changes in protein stability from point mutations without parameterization or system-specific customization, although further improvements should be possible with additional sampling and a better representation of the unfolded state of the protein. Here, we describe the FEP+ method as applied to protein stability calculations, summarize the large-scale retrospective validation results, and discuss limitations of the method, along with future directions for further improvements., (Copyright © 2016 Elsevier Ltd. All rights reserved.)