Modulation of Structure and Dynamics of Cardiac Troponin by Phosphorylation and Mutations Revealed by Molecular Dynamics Simulations.

Adrenaline acts on ß1 receptors in the heart muscle to enhance contractility, increase the heart rate, and increase the rate of relaxation (lusitropy) via activation of the cyclic AMP-dependent protein kinase, PKA. Phosphorylation of serines 22 and 23 in the N-terminal peptide of cardiac troponin I is responsible for lusitropy. Mutations associated with cardiomyopathy suppress the phosphorylation-dependent change. Key parts of troponin responsible for this modulatory system are disordered and cannot be resolved by conventional structural approaches. We performed all-atom molecular dynamics simulations (5 × 1.5 µs runs) of the troponin core (419 amino acids) in the presence of Ca2+ in the bisphosphorylated and unphosphorylated states for both wild-type troponin and the troponin C (cTnC) G159D mutant. PKA phosphorylation affects troponin dynamics. There is significant rigidification of the structure involving rearrangement of the cTnI(1-33)-cTnC interaction and changes in the distribution of the cTnC helix A/B angle, troponin I (cTnI) switch peptide (149-164) docking, and the angle between the regulatory head and ITC arm domains. The familial dilated cardiomyopathy cTnC G159D mutation whose Ca2+ sensitivity is not modulated by cTnI phosphorylation exhibits a structure inherently more rigid than the wild type, with phosphorylation reversing the direction of all metrics relative to the wild type.

Kinetics and Mechanisms of Hydrothermal Dehydration of Cyclic 1,2- and 1,4-Diols.

Hydrothermal dehydration is an attractive method for deoxygenation and upgrading of biofuels because it requires no reagents or catalysts other than superheated water. Although mono-alcohols cleanly deoxygenate via dehydration under many conditions, polyols such as those derived from saccharides and related structures are known to be recalcitrant with respect to dehydration. Here, we describe detailed mechanistic and kinetic studies of hydrothermal dehydration of 1,2- and 1,4-cyclohexanediols as model compounds to investigate how interactions between the hydroxyls can control the reaction. The diols generally dehydrate more slowly and have more complex reaction pathways than simple cyclohexanol. Although hydrogen bonding between hydroxyls is an important feature of the diol reactions, hydrogen bonding on its own does not explain the reduced reactivity. Rather, it is the way that hydrogen bonding influences the balance between the E1 and E2 elimination mechanisms. We also describe the reaction pathways and follow-up secondary reactions for the slower-dehydrating diols.

Large-Scale Distributed Training of Transformers for Chemical Fingerprinting.

Transformer models have become a popular choice for various machine learning tasks due to their often outstanding performance. Recently, transformers have been used in chemistry for classifying reactions, reaction prediction, physiochemical property prediction, and more. These models require huge amounts of data and localized compute to train effectively. In this work, we demonstrate that these models can successfully be trained for chemical problems in a distributed manner across many computers─a more common scenario for chemistry institutions. We introduce MFBERT: Molecular Fingerprints through Bidirectional Encoder Representations from Transformers. We use distributed computing to pre-train a transformer model on one of the largest aggregate datasets in chemical literature and achieve state-of-the-art scores on a virtual screening benchmark for molecular fingerprints. We then fine-tune our model on smaller, more specific datasets to generate more targeted fingerprints and assess their quality. We utilize a SentencePiece tokenization model, where the whole procedure from raw molecular representation to molecular fingerprints becomes data-driven, with no explicit tokenization rules.

Modulation of cardiac thin filament structure by phosphorylated troponin-I analyzed by protein-protein docking and molecular dynamics simulation.

Tropomyosin, controlled by troponin-linked Ca2+-binding, regulates muscle contraction by a macromolecular scale steric-mechanism that governs myosin-crossbridge-actin interactions. At low-Ca2+, C-terminal domains of troponin-I (TnI) trap tropomyosin in a position on thin filaments that interferes with myosin-binding, thus causing muscle relaxation. Steric inhibition is reversed at high-Ca2+ when TnI releases from F-actin-tropomyosin as Ca2+ and the TnI switch-peptide bind to the N-lobe of troponin-C (TnC). The opposite end of cardiac TnI contains a phosphorylation-sensitive ∼30 residue-long N-terminal peptide that is absent in skeletal muscle, and likely modifies these interactions in hearts. Here, PKA-dependent phosphorylation of serine 23 and 24 modulates Ca2+ and possibly switch-peptide binding to TnC, causing faster relaxation during the cardiac-cycle (lusitropy). The cardiac-specific N-terminal TnI domain is not captured in crystal structures of troponin or in cryo-EM reconstructions of thin filaments; thus, its global impact on thin filament structure and function is uncertain. Here, we used protein-protein docking and molecular dynamics simulation-based protocols to build a troponin model that was guided by and hence consistent with the recent seminal Yamada structure of Ca2+-activated thin filaments. We find that when present on thin filaments, phosphorylated Ser23/24 along with adjacent polar TnI residues interact closely with both tropomyosin and the N-lobe of TnC during our simulations. These interactions would likely bias tropomyosin to an off-state positioning on actin. In situ, such enhanced relaxation kinetics would promote cardiac lusitropy.

Lipid21: Complex Lipid Membrane Simulations with AMBER.

We extend the modular AMBER lipid force field to include anionic lipids, polyunsaturated fatty acid (PUFA) lipids, and sphingomyelin, allowing the simulation of realistic cell membrane lipid compositions, including raft-like domains. Head group torsion parameters are revised, resulting in improved agreement with NMR order parameters, and hydrocarbon chain parameters are updated, providing a better match with phase transition temperature. Extensive validation runs (0.9 µs per lipid type) show good agreement with experimental measurements. Furthermore, the simulation of raft-like bilayers demonstrates the perturbing effect of increasing PUFA concentrations on cholesterol molecules. The force field derivation is consistent with the AMBER philosophy, meaning it can be easily mixed with protein, small molecule, nucleic acid, and carbohydrate force fields.

Design, synthesis and evaluation of a tripodal receptor for phosphatidylinositol phosphates.

Phosphatidylinositol phosphates (PIPs) are membrane phospholipids that play crucial roles in a wide range of cellular processes. Their function is dictated by the number and positions of the phosphate groups in the inositol ring (with seven different PIPs being active in the cell). Therefore, there is significant interest in developing small-molecule receptors that can bind selectively to these species and in doing so affect their cellular function or be the basis for molecular probes. However, to date there are very few examples of such molecular receptors. Towards this aim, herein we report a novel tripodal molecule that acts as receptor for mono- and bis-phosphorylated PIPs in a cell free environment. To assess their affinity to PIPs we have developed a new cell free assay based on the ability of the receptor to prevent alkaline phosphatase from hydrolysing these substrates. The new receptor displays selectivity towards two out of the seven PIPs, namely PI(3)P and PI(3,4)P2. To rationalise these results, a DFT computational study was performed which corroborated the experimental results and provided insight into the host-guest binding mode.

A TDDFT investigation of the Photosystem II reaction center: Insights into the precursors to charge separation.

Photosystem II (PS II) captures solar energy and directs charge separation (CS) across the thylakoid membrane during photosynthesis. The highly oxidizing, charge-separated state generated within its reaction center (RC) drives water oxidation. Spectroscopic studies on PS II RCs are difficult to interpret due to large spectral congestion, necessitating modeling to elucidate key spectral features. Herein, we present results from time-dependent density functional theory (TDDFT) calculations on the largest PS II RC model reported to date. This model explicitly includes six RC chromophores and both the chlorin phytol chains and the amino acid residues <6 Å from the pigments' porphyrin ring centers. Comparing our wild-type model results with calculations on mutant D1-His-198-Ala and D2-His-197-Ala RCs, our simulated absorption-difference spectra reproduce experimentally observed shifts in known chlorophyll absorption bands, demonstrating the predictive capabilities of this model. We find that inclusion of both nearby residues and phytol chains is necessary to reproduce this behavior. Our calculations provide a unique opportunity to observe the molecular orbitals that contribute to the excited states that are precursors to CS. Strikingly, we observe two high oscillator strength, low-lying states, in which molecular orbitals are delocalized over ChlD1 and PheD1 as well as one weaker oscillator strength state with molecular orbitals delocalized over the P chlorophylls. Both these configurations are a match for previously identified exciton-charge transfer states (ChlD1+PheD1-)* and (PD2+PD1-)*. Our results demonstrate the power of TDDFT as a tool, for studies of natural photosynthesis, or indeed future studies of artificial photosynthetic complexes.

Understanding the Solvent Contribution to Chemical Reaction Barriers.

Absolute rate theories attempt to predict the rate constants of reactions from basic principles and independent data. For the contribution of solvent to a reaction rate constant, this requires connecting absolute rate data to fundamental solvent properties such as dielectric constant and refractive index. We have explored this connection for the unimolecular fragmentation reaction of a pinacol radical cation. The rate constants for fragmentation were measured as a function of temperature in 12 different solvents with dielectric constants from 4.7 to 36.2, and the free energies of activation for bond fragmentation in each solvent determined using transition state theory. Using the solvent effects on electron-transfer reactions as a starting point, Marcus theory was used to model the solvent effect on the reaction activation energies. The solvent contribution to both the activation free energy and the overall reaction energy is best described using the Born model rather than the Pekar solvation model. The solvent reorganization energies for bond fragmentation are substantially larger than solvent reorganization energies for electron transfer, presumably because of the requirement to translate the solvent molecules in the course of bond breaking.

Mechanism of Photoinduced Triplet Intermolecular Hydrogen Transfer between Cycloxydim and Chlorothalonil.

The possible reaction mechanisms for the experimentally observed hydrogen transfer between the herbicide cycloxydim (CD) and the triplet fungicide chlorothalonil (CT) were identified with density functional theory (DFT) and time-dependent density function theory (TDDFT) computations. Excited energy transfer (EET) calculations indicate that reactants for intermolecular hydrogen transfer were formed via energy transfer from triplet CT to ground state CD. Three possible reaction pathways after EET were identified, and hydrogen transfer from the hydroxyl group on the cyclohexane ring of CD to CT exhibited the lowest energy barrier. Natural population analysis (NPA) along the reaction pathways has confirmed that the pathways involved either electron transfer induced proton transfer or coupled electron-proton transfer, leading to different potential energy profiles. Electrostatic potential (ESP) study substantiated the reaction mechanisms in different pathways. This study suggests an explanation for the accelerated photodegradation of CD by CT and provides a pipeline for future studies of photoinduced intermolecular hydrogen transfer.

Imaging plasma membrane phase behaviour in live cells using a thiophene-based molecular rotor.

Molecular rotors have emerged as versatile probes of microscopic viscosity in lipid bilayers, although it has proved difficult to find probes that stain both phases equally in phase-separated bilayers. Here, we investigate the use of a membrane-targeting viscosity-sensitive fluorophore based on a thiophene moiety with equal affinity for ordered and disordered lipid domains to probe ordering and viscosity within artificial lipid bilayers and live cell plasma membranes.

Molecular dynamics studies on the DNA-binding process of ERG.

The ETS family of transcription factors regulate gene targets by binding to a core GGAA DNA-sequence. The ETS factor ERG is required for homeostasis and lineage-specific functions in endothelial cells, some subset of haemopoietic cells and chondrocytes; its ectopic expression is linked to oncogenesis in multiple tissues. To date details of the DNA-binding process of ERG including DNA-sequence recognition outside the core GGAA-sequence are largely unknown. We combined available structural and experimental data to perform molecular dynamics simulations to study the DNA-binding process of ERG. In particular we were able to reproduce the ERG DNA-complex with a DNA-binding simulation starting in an unbound configuration with a final root-mean-square-deviation (RMSD) of 2.1 Å to the core ETS domain DNA-complex crystal structure. This allowed us to elucidate the relevance of amino acids involved in the formation of the ERG DNA-complex and to identify Arg385 as a novel key residue in the DNA-binding process. Moreover we were able to show that water-mediated hydrogen bonds are present between ERG and DNA in our simulations and that those interactions have the potential to achieve sequence recognition outside the GGAA core DNA-sequence. The methodology employed in this study shows the promising capabilities of modern molecular dynamics simulations in the field of protein DNA-interactions.

Troponin structure: its modulation by Ca(2+) and phosphorylation studied by molecular dynamics simulations.

The only available crystal structure of the human cardiac troponin molecule (cTn) in the Ca(2+) activated state does not include crucial segments, including the N-terminus of the cTn inhibitory subunit (cTnI). We have applied all-atom molecular dynamics (MD) simulations to study the structure and dynamics of cTn, both in the unphosphorylated and bis-phosphorylated states at Ser23/Ser24 of cTnI. We performed multiple microsecond MD simulations of wild type (WT) cTn (6, 5 µs) and bisphosphorylated (SP23/SP24) cTn (9 µs) on a 419 amino acid cTn model containing human sequence cTnC (1-161), cTnI (1-171) and cTnT (212-298), including residues not present in the crystal structure. We have compared our results to previous computational studies, and proven that longer simulations and a water box of at least 25 Å are needed to sample the interesting conformational shifts both in the native and bis-phosphorylated states. As a consequence of the introduction into the model of the C-terminus of cTnT that was missing in previous studies, cTnC-cTnI interactions that are responsible for the cTn dynamics are altered. We have also shown that phosphorylation does not increase cTn fluctuations, and its effects on the protein-protein interaction profiles cannot be assessed in a significant way. Finally, we propose that phosphorylation could provoke a loss of Ca(2+) by stabilizing out-of-coordination distances of the cTnC's EF hand II residues, and in particular Ser 69.

Simulation of lipid bilayer self-assembly using all-atom lipid force fields.

In this manuscript we expand significantly on our earlier communication by investigating the bilayer self-assembly of eight different types of phospholipids in unbiased molecular dynamics (MD) simulations using three widely used all-atom lipid force fields. Irrespective of the underlying force field, the lipids are shown to spontaneously form stable lamellar bilayer structures within 1 microsecond, the majority of which display properties in satisfactory agreement with the experimental data. The lipids self-assemble via the same general mechanism, though at formation rates that differ both between lipid types, force fields and even repeats on the same lipid/force field combination. In addition to zwitterionic phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids, anionic phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids are represented. To our knowledge this is the first time bilayer self-assembly of phospholipids with negatively charged head groups is demonstrated in all-atom MD simulations.

Reversible Electrochemical Trapping of Carbon Dioxide Using 4,4'-Bipyridine That Does Not Require Thermal Activation.

Sequestering carbon dioxide emissions by the trap and release of CO2 via thermally activated chemical reactions has proven problematic because of the energetic requirements of the release reactions. Here we demonstrate trap and release of carbon dioxide using electrochemical activation, where the reactions in both directions are exergonic and proceed rapidly with low activation barriers. One-electron reduction of 4,4'-bipyridine forms the radical anion, which undergoes rapid covalent bond formation with carbon dioxide to form an adduct. One-electron oxidation of this adduct releases the bipyridine and carbon dioxide. Reversible trap and release of carbon dioxide over multiple cycles is demonstrated in solution at room temperature, and without the requirement for thermal activation.

Organic Oxidations Using Geomimicry.

Oxidations of phenylacetic acid to benzaldehyde, benzyl alcohol to benzaldehyde, and benzaldehyde to benzoic acid have been observed, in water as the solvent and using only copper(II) chloride as the oxidant. The reactions are performed at 250 °C and 40 bar, conditions that mimic hydrothermal reactions that are geochemically relevant. Speciation calculations show that the oxidizing agent is not freely solvated copper(II) ions, but complexes of copper(II) with chloride and carboxylate anions. Measurements of the reaction stoichiometries and also of substituent effects on reactivity allow plausible mechanisms to be proposed. These oxidation reactions are relevant to green chemistry in that they proceed in high chemical yield in water as the solvent and avoid the use of toxic heavy metal oxidizing reagents.

A Parameterization of Cholesterol for Mixed Lipid Bilayer Simulation within the Amber Lipid14 Force Field.

The Amber Lipid14 force field is expanded to include cholesterol parameters for all-atom cholesterol and lipid bilayer molecular dynamics simulations. The General Amber and Lipid14 force fields are used as a basis for assigning atom types and basic parameters. A new RESP charge derivation for cholesterol is presented, and tail parameters are adapted from Lipid14 alkane tails. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers are simulated at a range of cholesterol contents. Experimental bilayer structural properties are compared with bilayer simulations and are found to be in good agreement. With this parameterization, another component of complex membranes is available for molecular dynamics with the Amber Lipid14 force field.

Imaging phase separation in model lipid membranes through the use of BODIPY based molecular rotors.

In order to fully understand the dynamics of processes within biological lipid membranes, it is necessary to possess an intimate knowledge of the physical state and ordering of lipids within the membrane. Here we report the use of three molecular rotors based on meso-substituted boron-dipyrrin (BODIPY) in combination with fluorescence lifetime spectroscopy to investigate the viscosity and phase behaviour of model lipid bilayers. In phase-separated giant unilamellar vesicles, we visualise both liquid-ordered (Lo) and liquid-disordered (Ld) phases using fluorescence lifetime imaging microscopy (FLIM), determining their associated viscosity values, and investigate the effect of composition on the viscosity of these phases. Additionally, we use molecular dynamics simulations to investigate the orientation of the BODIPY probes within the bilayer, as well as using molecular dynamics simulations and fluorescence correlation spectroscopy (FCS) to compare diffusion coefficients with those predicted from the fluorescence lifetimes of the probes.

All-atom lipid bilayer self-assembly with the AMBER and CHARMM lipid force fields.

This communication reports the first example of spontaneous lipid bilayer formation in unbiased all-atom molecular dynamics (MD) simulations. Using two different lipid force fields we show simulations started from random mixtures of lipids and water in which four different types of phospholipids self-assemble into organized bilayers in under 1 microsecond.

Sphalerite is a geochemical catalyst for carbon-hydrogen bond activation.

Reactions among minerals and organic compounds in hydrothermal systems are critical components of the Earth's deep carbon cycle, provide energy for the deep biosphere, and may have implications for the origins of life. However, there is limited information as to how specific minerals influence the reactivity of organic compounds. Here we demonstrate mineral catalysis of the most fundamental component of an organic reaction: the breaking and making of a covalent bond. In the absence of mineral, hydrothermal reaction of cis- and trans-1,2-dimethylcyclohexane is extremely slow and generates many products. In the presence of sphalerite (ZnS), however, the reaction rate increases dramatically and one major product is formed: the corresponding stereoisomer. Isotope studies show that the sphalerite acts as a highly specific heterogeneous catalyst for activation of a single carbon-hydrogen bond in the dimethylcyclohexanes.

Hydrothermal photochemistry as a mechanistic tool in organic geochemistry: the chemistry of dibenzyl ketone.

Hydrothermal organic transformations under geochemically relevant conditions can result in complex product mixtures that form via multiple reaction pathways. The hydrothermal decomposition reactions of the model ketone dibenzyl ketone form a mixture of reduction, dehydration, fragmentation, and coupling products that suggest simultaneous and competitive radical and ionic reaction pathways. Here we show how Norrish Type I photocleavage of dibenzyl ketone can be used to independently generate the benzyl radicals previously proposed as the primary intermediates for the pure hydrothermal reaction. Under hydrothermal conditions, the benzyl radicals undergo hydrogen atom abstraction from dibenzyl ketone and para-coupling reactions that are not observed under ambient conditions. The photochemical method allows the primary radical coupling products to be identified, and because these products are generated rapidly, the method also allows the kinetics of the subsequent dehydration and Paal-Knorr cyclization reactions to be measured. In this way, the radical and ionic thermal and hydrothermal reaction pathways can be studied separately.