An efficient variational method is presented for estimating the diffusion coefficients and free-energy profiles along selected collective variables from projected molecular dynamics trajectories under both equilibrium and non-equilibrium conditions. This method is based on the assumption that the short-time transition probability density of the coordinate moves can be approximated by a Gaussian form. Defining a loss function as the sum of Kullback-Leibler divergences between the analytical short-time propagators of an overdamped Langevin model and those estimated directly from the projected trajectories maximizes the agreement between the two and allows for its analytic evaluation. For cases where the Gaussian approximation is insufficient, we present a robust alternative. To efficiently minimize this loss function by varying diffusion and free-energy profiles along collective variables, we use an adaptive Monte Carlo scheme. The method is applied to two model systems exhibiting diffusive dynamics, as well as to water diffusion across the interface of a biomolecular condensate, demonstrating its robustness and accuracy.

The use of protein nanopores has proven to be very promising for the identification of proteins or the sequencing of peptides. However, their widespread use in biosensor applications is limited by more complex molecular properties of proteins in comparison to nucleic acids. Here, we developed an α-helical Fav nanopore from the coral Orbicella faveolata to detect human histones using a commercial MinION device. The engineered nanopores showed stable insertion into the polymeric membrane support. Bulk analysis of several tens to hundreds of pores per measurement revealed significant differences in mean current passing the pore and current noise resulting from capturing different full-length human histones or their post-translationally modified variants into the pore lumen. Importantly, detailed blocking analyses confirmed histone-specific signals that allow further discrimination between two medically important extracellular histones in mixtures. The newly developed Fav nanopore and high-throughput approach for the detection of biomedically important proteins is an important step towards the widespread use of nanopores in modern analytics.

The structure and function of membrane proteins depend on their interactions with lipids that constitute membranes. Actinoporins are α-pore-forming proteins that bind preferentially to sphingomyelin-containing membranes, where they oligomerize and form transmembrane pores. Through a comprehensive cryo-electron microscopic analysis of a pore formed by an actinoporin Fav from the coral Orbicella faveolata, we show that the octameric pore interacts with 112 lipids in the upper leaflet of the membrane, reveal the roles of lipids, and demonstrate that the actinoporin surface is suited for binding multiple receptor sphingomyelin molecules. When cholesterol is present in the membrane, it forms a cluster of four molecules associated with each protomer. Atomistic simulations support the structural data and reveal additional effects of the pore on the lipid membrane. These data reveal a complex network of protein-lipid and lipid-lipid interactions and an underrated role of lipids in the structure and function of transmembrane protein complexes. The structure and function of membrane proteins depend on lipid interactions. Cryo-EM analysis of the actinoporin Fav pore reveals octameric structure binding 112 lipids and clusters of cholesterols. Molecular simulations highlight complex protein-lipid interactions crucial for structure.

Muramyl ligases are multidomain enzymes involved in intracellular steps of bacterial peptidoglycan synthesis and are considered promising targets for the development of new antibacterial agents. Among them, Mur ligase D (MurD) has been most widely used for structure-based design, but success has been limited. Here, we determine the 15N NMR spin relaxation parameters of apo and bound states of MurD in solution. We introduce a principal component analysis of the spectral densities derived from the NMR relaxation data, which provides a mechanistic insight into the dynamic events at the residue level. Compensation effects (ps-ms timescale) and conformational exchange dynamics (µs-ms timescale), the latter also measured independently, were revealed in bound and unbound MurD, which should be considered in the design of structurally novel Mur inhibitors. The mechanistic consideration used in our study can be broadly applicable to other systems for deciphering their specific dynamic mechanisms. Mur ligase D (MurD) is a promising antibacterial target, but its dynamics remain underexplored. Here, the authors use 15N nuclear magnetic resonance to probe apo and bound states of MurD in solution, showing that dynamic events on a time scale of picoseconds to milliseconds have to be considered in the design of structurally novel Mur inhibitors.

The structural diversity and tunability of the capsid proteins (CPs) of various icosahedral and rod-shaped viruses have been well studied and exploited in the development of smart hybrid nanoparticles. However, the potential of CPs of the wide-spread flexuous filamentous plant viruses remains to be explored. Here, we show that we can control the shape, size, RNA encapsidation ability, symmetry, stability and surface functionalization of nanoparticles through structure-based design of CP from potato virus Y (PVY). We provide high-resolution insight into CP-based self-assemblies, ranging from large polymorphic or monomorphic filaments to smaller annular, cubic or spherical particles. Furthermore, we show that we can prevent CP self-assembly in bacteria by fusion with a cleavable protein, enabling controlled nanoparticle formation in vitro. Understanding the remarkable structural diversity of PVY CP not only provides possibilities for the production of biodegradable nanoparticles, but may also advance future studies of CP’s polymorphism in a biological context.

The structure and function of membrane proteins depend on their interactions with the lipid molecules that constitute lipid membranes. Actinoporins are a family of α-pore-forming proteins that bind specifically to sphingomyelin-containing lipid membranes, where they oligomerize and form transmembrane pores. The numerous contacts they form with the lipid membrane make them an exemplary object for studying the different roles that lipids play in the structure and function of membrane proteins. Through a comprehensive cryo-electron microscopic analysis of a pore formed by an actinoporin Fav from the coral Orbicella faveolata , we show that the octameric pore interacts with 112 lipids in the upper leaflet of the membrane. The structures of Fav pores formed on different lipid membranes reveal the different roles of lipids and demonstrate that the actinoporin surface is perfectly suited for binding multiple receptor sphingomyelin molecules. When cholesterol is present in the membrane, it forms nanodomains associated with the pore, leading to a tighter arrangement of lipids, which in turn increases the stability of the pores. Atomistic simulations support the structural data, show that the protein-bound lipids are not mobile, and reveal additional effects of the pore on the lipid membrane. Overall, these data reveal a complex network of protein-lipid and lipid-lipid interactions, and an underrated role of lipids in the structure and function of transmembrane protein complexes. ### Competing Interest Statement The authors have declared no competing interest.

Thiazolin-4-ones and their derivatives represent important heterocyclic scaffolds with various applications in medicinal chemistry. For that reason, the synthesis of two 5-substituted thiazolidin-4-one derivatives was performed. Their structure assignment was conducted by NMR experiments (2D-COSY, 2D-NOESY, 2D-HSQC and 2D-HMBC) and conformational analysis was conducted through Density Functional Theory calculations and 2D-NOESY. Conformational analysis showed that these two molecules adopt exo conformation. Their global minimum structures have two double bonds (C=N, C=C) in Z conformation and the third double (C=N) in E. Our DFT results are in agreement with the 2D-NMR measurements. Furthermore, the reaction isomerization paths were studied via DFT to check the stability of the conformers. Finally, some potential targets were found through the SwissADME platform and docking experiments were performed. Both compounds bind strongly to five macromolecules (triazoloquinazolines, mglur3, Jak3, Danio rerio HDAC6 CD2, acetylcholinesterase) and via SwissADME it was found that these two molecules obey Lipinski’s Rule of Five.

This work presents a study on the optical and mechanical degradation of parabolic trough collector absorber coatings produced through the spray coating application technique of in-house developed paint. The main aim of this investigation is to prepare, cure, load, and analyze the absorber coating on the substrate under conditions that mimic the on-field thermal properties. This research incorporates predicted isothermal and cyclic loads for parabolic trough systems as stresses. Biweekly inspections of loaded, identical samples monitored the degradation process. We further used the cascade of data from optical, oxide-thickening, crack length, and pull-off force measurements in mathematical modelling to predict the service life of the parabolic trough collector. The results collected and used in modelling suggested that cyclic load in combination with iso-thermal load is responsible for coating fatigue, influencing the solar absorber optical values and resulting in lower energy transformation efficiency. Finally, easy-to-apply coatings made out of spinel-structured black pigment and durable binder could serve as a low-cost absorber coating replacement for a new generation of parabolic trough collectors, making it possible to harvest solar energy to provide medium-temperature heat to decarbonize future food, tobacco, and paint production industrial processes.

The human sodium–glucose cotransporter protein (SGLT1) is an important representative of the sodium solute symporters belonging to the secondary active transporters that are critical to the homeostasis of sugar, sodium, and water in the cell. The underlying transport mechanism of SGLT1 is based on switching between inward- and outward-facing conformations, known as the alternating access model, which is crucial for substrate transport, and has also been postulated for water permeation. However, the nature of water transport remains unclear and is disputed along the passive and active transport, with the latter postulating the presence of the pumping effect. To better examine the water transport in SGLT1, we performed a series of equilibrium all-atom molecular dynamics simulations, totaling over 6 μs of sample representative conformational states of SGLT1 and its complexes, with the natural substrates, ions, and inhibitors. In addition to elucidating the basic physical factors influencing water permeation, such as channel openings and energetics, we focus on dynamic flexibility and its relationship with domain motion. Our results clearly demonstrate a dependence of instantaneous water flux on the channel opening and local water diffusion in the channel, strongly supporting the existence of a passive water transport in SGLT1. In addition, a strong correlation found between the local water diffusion and protein domain motion, resembling the “rocking-bundle” motion, reveals its facilitating role in the water transport.

Sodium glucose cotransporters (SGLTs) are cotransporters located in the cell membrane of various epithelia that uptake glucose or galactose and sodium into the cell. Its founding member, SGLT1, represents a major pharmaceutically relevant target protein for development of new antidiabetic drugs, in addition to being the target protein of the oral rehydration therapy. Previous studies focused primarily on the transport of substrates and ions, while our study focuses on the effect of water transport. SGLT1 is implicated in the absorption of water, yet the exact mechanism of how the water absorption occurs or how inhibitors of SGLT1, such as phlorizin, are able to inhibit it is still unclear. Here we present a comprehensive study based on molecular dynamics simulations with the aim of determining the influence of the energetic and dynamic properties of SGLT1, which are influenced by selected sugar uptake inhibitors on water permeation.

Linde type A (LTA) aluminophosphate is a promising candidate for an energy storage material used for low-temperature solar and waste-heat management. The mechanism of reversible water adsorption, which is the basis for potential industrial applications, is still not clear. In this paper, we provide mechanistic insight into various aspects of the hydration process using molecular modeling methods. Building on accurate DFT calculations and available experimental data, we first refine the existing empirical force-field used in subsequent classical molecular dynamics simulations that captures the relevant physics of the water binding process. We succeed in fully reproducing the experimentally determined X-ray structure factors and use them to estimate the number of water molecules present in the fully hydrated state of the material. Furthermore, we show that the translational and orientational mobility of the confined water is significantly reduced and resembles the dynamics of glassy systems.

The application of terahertz radiation has been shown to affect both protein structure and cellular function. As the key to such structural changes lies in the dynamic response of a protein, we focus on the susceptibility of the protein’s internal dynamics to mechanical stress induced by acoustic pressure waves. We use the open-boundary molecular dynamics method, which allows us to simulate the protein exposed to acoustic waves. By analyzing the dynamic fluctuations of the protein subunits, we demonstrate that the protein is highly susceptible to acoustic waves with frequencies corresponding to those of the internal protein vibrations. This is confirmed by changes in the compactness of the protein structure. As the amplitude of the pressure wave increases, even larger deviations from average positions and larger changes in protein compactness are observed. Furthermore, performing the mode-projection analysis, we show that the breathing-like character of collective modes is enhanced at frequencies corresponding to those used to excite the protein.

Proteins are natural polymers that play an essential role in both living organisms and biotechnological applications. During certain bioprocessing steps, they can be exposed to significant mechanical stress induced by, for example, shear flow or sonication, resulting in reduced therapeutic efficacy, aggregation, or even a loss of activity. For this reason, there is a need to understand and determine the susceptibility of the protein activity to the experienced mechanical stress. To acquire this knowledge, it is necessary to study the rotational dynamics of the protein. Commonly, the rotational dynamics of soft molecules is interpreted based on a theoretical analysis performed in an inertial laboratory frame. However, the obtained angular velocity mixes pure rotations and vibrations with angular momentum, consequently lacking a clear dynamical interpretation. On the other hand, the use of the noninertial internal Eckart frame allows the determination of pure angular velocity as it minimizes the coupling between the rotational and vibrational degrees of freedom. In the present work, by conducting open-boundary molecular dynamics simulations and exploiting the Eckart frame formalism, we study the rotational dynamics of a small protein under the shear flow of various strengths. Our results show that the angular velocity increases nonlinearly with increasing shear rate. Furthermore, the protein gains vibrational angular momentum at higher shear rates, which is reflected in the higher angular velocity computed by employing the Eckart frame formalism and confirmed by analysis of the contributions to the total kinetic energy of the biomolecule.

A new method for efficient and robust determination of thermal conductivity in liquids is presented. The method is based on the search for an optimal agreement between the analytical solution of the heat transfer in a continuum model described by Fourier law and the relaxation of thermal inhomogeneity simulated by nonequilibrium MD simulations. Our approach exploits a transient regime in which a sys-tem relaxes towards equilibrium after the introduction of a small temperature perturbation in a spatially confined part of the system. The applicability of this new method is demonstrated on liquid argon and two water models, the mW and SPC models. & COPY; 2022 Elsevier B.V. All rights reserved.

Perforin is a pore-forming protein that plays a crucial role in the immune system by clearing virus-infected or tumor cells. It is released from cytotoxic granules of immune cells and forms pores in targeted lipid membranes to deliver apoptosis-inducing granzymes. It is a very cytotoxic protein and is therefore adapted not to act in producing cells. Its activity is regulated by the requirement for calcium ions for optimal activity. However, the exact affinity of perforin for calcium ions has not yet been determined. We conducted a molecular dynamics simulation in the absence or presence of calcium ions that showed that binding of at least three calcium ions is required for stable perforin binding to the lipid membrane. Biophysical studies using surface plasmon resonance and microscale thermophoresis were then performed to estimate the binding affinities of native human and recombinant mouse perforin for calcium ions. Both approaches showed that mouse perforin has a several fold higher affinity for calcium ions than that of human perforin. This was attributed to a particular residue, tryptophan at position 488 in mouse perforin, which is replaced by arginine in human perforin. This represents an additional mechanism to control the activity of human perforin.

Pore-forming toxins are an important component of the venom of many animals. Actinoporins are potent cytolysins that were first detected in the venom of sea anemones; however, they are occasionally found in animals other than cnidarians and are expanded in a few predatory gastropods. Here, we report the presence of 27 unique actinoporin-like genes with monophyletic origin in Mytilus galloprovincialis, which we have termed mytiporins. These mytiporins exhibited a remarkable level of molecular diversity and gene presence-absence variation, which warranted further studies aimed at elucidating their functional role. We structurally and functionally characterized mytiporin-1 and found significant differences from the archetypal actinoporin fragaceatoxin C. Mytiporin-1 showed weaker permeabilization activity, no specificity towards sphingomyelin, and weak activity in model lipid systems with negatively charged lipids. In contrast to fragaceatoxin C, which forms octameric pores, functional mytiporin-1 pores on negatively charged lipid membranes were hexameric. Similar hexameric pores were observed for coluporin-26 from Cumia reticulata and a conoporin from Conus andremenezi. This indicates that also other molluscan actinoporin-like proteins differ from fragaceatoxin C. Although the functional role of mytiporins in the context of molluscan physiology remains to be elucidated, the lineage-specific gene family expansion event that characterizes mytiporins indicates that strong selective forces acted on their molecular diversification. Given the tissue distribution of mytiporins, this process may have broadened the taxonomic breadth of their biological targets, which would have important implications for digestive processes or mucosal immunity.

The KV10.1 voltage-gated potassium channel is highly expressed in 70% of tumors, and thus represents a promising target for anticancer drug discovery. However, only a few ligands are known to inhibit KV10.1, and almost all also inhibit the very similar cardiac hERG channel, which can lead to undesirable side-effects. In the absence of the structure of the KV10.1–inhibitor complex, there remains the need for new strategies to identify selective KV10.1 inhibitors and to understand the binding modes of the known KV10.1 inhibitors. To investigate these binding modes in the central cavity of KV10.1, a unique approach was used that allows derivation and analysis of ligand–protein interactions from molecular dynamics trajectories through pharmacophore modeling. The final molecular dynamics-derived structure-based pharmacophore model for the simulated KV10.1–ligand complexes describes the necessary pharmacophore features for KV10.1 inhibition and is highly similar to the previously reported ligand-based hERG pharmacophore model used to explain the nonselectivity of KV10.1 pore blockers. Moreover, analysis of the molecular dynamics trajectories revealed disruption of the π–π network of aromatic residues F359, Y464, and F468 of KV10.1, which has been reported to be important for binding of various ligands for both KV10.1 and hERG channels. These data indicate that targeting the KV10.1 channel pore is also likely to result in undesired hERG inhibition, and other potential binding sites should be explored to develop true KV10.1-selective inhibitors as new anticancer agents.