Localization of the tubby domain, a PI(4,5)P2 biosensor, to E-Syt3-rich endoplasmic reticulum-plasma membrane junctions.

The phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] acts as a signaling lipid at the plasma membrane (PM) with pleiotropic regulatory actions on multiple cellular processes. Signaling specificity might result from spatiotemporal compartmentalization of the lipid and from combinatorial binding of PI(4,5)P2 effector proteins to additional membrane components. Here, we analyzed the spatial distribution of tubbyCT, a paradigmatic PI(4,5)P2-binding domain, in live mammalian cells by total internal reflection fluorescence (TIRF) microscopy and molecular dynamics simulations. We found that unlike other well-characterized PI(4,5)P2 recognition domains, tubbyCT segregates into distinct domains within the PM. TubbyCT enrichment occurred at contact sites between PM and endoplasmic reticulum (ER) (i.e. at ER-PM junctions) as shown by colocalization with ER-PM markers. Localization to these sites was mediated in a combinatorial manner by binding to PI(4,5)P2 and by interaction with a cytosolic domain of extended synaptotagmin 3 (E-Syt3), but not other E-Syt isoforms. Selective localization to these structures suggests that tubbyCT is a novel selective reporter for a ER-PM junctional pool of PI(4,5)P2. Finally, we found that association with ER-PM junctions is a conserved feature of tubby-like proteins (TULPs), suggesting an as-yet-unknown function of TULPs.

Martini 3: a general purpose force field for coarse-grained molecular dynamics.

The coarse-grained Martini force field is widely used in biomolecular simulations. Here we present the refined model, Martini 3 ( http://cgmartini.nl ), with an improved interaction balance, new bead types and expanded ability to include specific interactions representing, for example, hydrogen bonding and electronic polarizability. The updated model allows more accurate predictions of molecular packing and interactions in general, which is exemplified with a vast and diverse set of applications, ranging from oil/water partitioning and miscibility data to complex molecular systems, involving protein-protein and protein-lipid interactions and material science applications as ionic liquids and aedamers.

Perspective: a stirring role for metabolism in cells.

Based on recent findings indicating that metabolism might be governed by a limit on the rate at which cells can dissipate Gibbs energy, in this Perspective, we propose a new mechanism of how metabolic activity could globally regulate biomolecular processes in a cell. Specifically, we postulate that Gibbs energy released in metabolic reactions is used to perform work, allowing enzymes to self-propel or to break free from supramolecular structures. This catalysis-induced enzyme movement will result in increased intracellular motion, which in turn can compromise biomolecular functions. Once the increased intracellular motion has a detrimental effect on regulatory mechanisms, this will establish a feedback mechanism on metabolic activity, and result in the observed thermodynamic limit. While this proposed explanation for the identified upper rate limit on cellular Gibbs energy dissipation rate awaits experimental validation, it offers an intriguing perspective of how metabolic activity can globally affect biomolecular functions and will hopefully spark new research.

Protein dynamics and lipid affinity of monomeric, zeaxanthin-binding LHCII in thylakoid membranes.

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from 13C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.

Small ionic radii limit time step in Martini 3 molecular dynamics simulations.

Among other improvements, the Martini 3 coarse-grained force field provides a more accurate description of the solvation of protein pockets and channels through the consistent use of various bead types and sizes. Here, we show that the representation of Na+ and Cl- ions as "tiny" (TQ5) beads limits the accessible time step to 25 fs. By contrast, with Martini 2, time steps of 30-40 fs were possible for lipid bilayer systems without proteins. This limitation is relevant for systems that require long equilibration times. We derive a quantitative kinetic model of time-integration instabilities in molecular dynamics (MD) as a function of the time step, ion concentration and mass, system size, and simulation time. We demonstrate that ion-water interactions are the main source of instability at physiological conditions, followed closely by ion-ion interactions. We show that increasing the ionic masses makes it possible to use time steps up to 40 fs with minimal impact on static equilibrium properties and dynamical quantities, such as lipid and solvent diffusion coefficients. Increasing the size of the bead representing the ions (and thus changing their hydration) also permits longer time steps. For a soluble protein, we find that increasing the mass of tiny beads also on the protein permits simulations with 30-fs time steps. The use of larger time steps in Martini 3 results in a more efficient exploration of configuration space. The kinetic model of MD simulation crashes can be used to determine the maximum allowed time step upfront for an efficient use of resources and whenever sampling efficiency is critical.

Computational Prediction of ω-Transaminase Specificity by a Combination of Docking and Molecular Dynamics Simulations.

ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialis with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.

Molecular dynamics simulations in photosynthesis.

Photosynthesis is regulated by a dynamic interplay between proteins, enzymes, pigments, lipids, and cofactors that takes place on a large spatio-temporal scale. Molecular dynamics (MD) simulations provide a powerful toolkit to investigate dynamical processes in (bio)molecular ensembles from the (sub)picosecond to the (sub)millisecond regime and from the Å to hundreds of nm length scale. Therefore, MD is well suited to address a variety of questions arising in the field of photosynthesis research. In this review, we provide an introduction to the basic concepts of MD simulations, at atomistic and coarse-grained level of resolution. Furthermore, we discuss applications of MD simulations to model photosynthetic systems of different sizes and complexity and their connection to experimental observables. Finally, we provide a brief glance on which methods provide opportunities to capture phenomena beyond the applicability of classical MD.

Lipid Fingerprints and Cofactor Dynamics of Light-Harvesting Complex II in Different Membranes.

Plant light-harvesting complex II (LHCII) is the key antenna complex for plant photosynthesis. We present coarse-grained molecular dynamics simulations of monomeric and trimeric LHCII in a realistic thylakoid membrane environment based on the Martini force field. The coarse-grained protein model has been optimized with respect to atomistic reference simulations. Our simulations provide detailed insights in the thylakoid lipid fingerprint of LHCII which compares well with experimental data from membrane protein purification. Comparing the monomer and trimeric LHCII reveals a stabilizing effect of trimerization on the chromophores as well as the protein. Moreover, the average chromophore distance shortens in the trimer leading to stronger excitonic couplings. When changing the native thylakoid environment to a model membrane the protein flexibility remains constant, whereas the chromophore flexibility is reduced. Overall, the presented LHCII model lays the foundation to investigate the µs dynamics of this key antenna protein of plants.

Ultrafast Reactive Quantum Dynamics Coupled to Classical Solvent Dynamics Using an Ehrenfest Approach.

The inclusion of solvent effects in the theoretical analysis of molecular processes becomes increasingly important. Currently, it is not feasible to directly include the solvent on the quantum level. We use an Ehrenfest approach to study the coupled time evolution of quantum dynamically treated solutes and classical solvents system. The classical dynamics of the solvent is coupled to the wavepacket dynamics of the solute and rotational and translational degrees of freedom of the solute are included classically. This allows quantum dynamics simulations for ultrafast processes that are decided by environment interactions without explicit separation of time scales. We show the application to the dissociation of ICN in liquid Ar as a proof of principal system and to the more applied example of uracil in water.

Controlling Photorelaxation in Uracil with Shaped Laser Pulses: A Theoretical Assessment.

The RNA nucleobase uracil can suffer from photodamage when exposed to UV light, which may lead to severe biological defects. To prevent this from happening in most cases, uracil exhibits an ultrafast relaxation mechanism from the electronically excited state back to the ground state. In our theoretical work, we demonstrate how this process can be significantly influenced using shaped laser pulses. This not only sheds new light on how efficient nature is in preventing biologically momentous photodamage. We also show a way to entirely prevent photorelaxation by preparing a long-living wave packet in the excited state. This can enable new experiments dedicated to finding the photochemical pathways leading to uracil photodamage. The optimized laser pulses we present fulfill all requirements to be experimentally accessible.

Design of specially adapted reactive coordinates to economically compute potential and kinetic energy operators including geometry relaxation.

Quantum dynamics simulations require prior knowledge of the potential energy surface as well as the kinetic energy operator. Typically, they are evaluated in a low-dimensional subspace of the full configuration space of the molecule as its dimensionality increases proportional to the number of atoms. This entails the challenge to find the most suitable subspace. We present an approach to design specially adapted reactive coordinates spanning this subspace. In addition to the essential geometric changes, these coordinates take into account the relaxation of the non-reactive coordinates without the necessity of performing geometry optimizations at each grid point. The method is demonstrated for an ultrafast photoinduced bond cleavage in a commonly used organic precursor for the generation of electrophiles. The potential energy surfaces for the reaction as well as the Wilson G-matrix as part of the kinetic energy operator are shown for a complex chemical reaction, both including the relaxation of the non-reactive coordinates on equal footing. A microscopic interpretation of the shape of the G-matrix elements allows to analyze the impact of the non-reactive coordinates on the kinetic energy operator. Additionally, we compare quantum dynamics simulations with and without the relaxation of the non-reactive coordinates included in the kinetic energy operator to demonstrate its influence.

Ion-pairing of phosphonium salts in solution: C-H⋠⋠⋠halogen and C-H⋠⋠⋠π hydrogen bonds.

The (1) H NMR chemical shifts of the C(α)H protons of arylmethyl triphenylphosphonium ions in CD2 Cl2 solution strongly depend on the counteranions X(-) . The values for the benzhydryl derivatives Ph2 CHPPh3 (+) X(-) , for example, range from δH =8.25 (X(-) =Cl(-) ) over 6.23 (X(-) =BF4 (-) ) to 5.72 ppm (X(-) =BPh4 (-) ). Similar, albeit weaker, counterion-induced shifts are observed for the ortho-protons of all aryl groups. Concentration-dependent NMR studies show that the large shifts result from the deshielding of the protons by the anions, which decreases in the order Cl(-) > Br(-) ≫ BF4 (-) > SbF6 (-) . For the less bulky derivatives PhCH2 PPh3 (+) X(-) , we also find CH⋅⋅⋅Ph interactions between C(α)H and a phenyl group of the BPh4 (-) anion, which result in upfield NMR chemical shifts of the C(α)H protons. These interactions could also be observed in crystals of (p-CF3 -C6 H4 )CH2 PPh3 (+) BPh4 (-) . However, the dominant effects causing the counterion-induced shifts in the NMR spectra are the CH⋅⋅⋅X(-) hydrogen bonds between the phosphonium ion and anions, in particular Cl(-) or Br(-) . This observation contradicts earlier interpretations which assigned these shifts predominantly to the ring current of the BPh4 (-) anions. The concentration dependence of the (1) H NMR chemical shifts allowed us to determine the dissociation constants of the phosphonium salts in CD2 Cl2 solution. The cation-anion interactions increase with the acidity of the C(α)H protons and the basicity of the anion. The existence of CH⋅⋅⋅X(-) hydrogen bonds between the cations and anions is confirmed by quantum chemical calculations of the ion pair structures, as well as by X-ray analyses of the crystals. The IR spectra of the Cl(-) and Br(-) salts in CD2 Cl2 solution show strong red-shifts of the CH stretch bands. The CH stretch bands of the tetrafluoroborate salt PhCH2 PPh3 (+) BF4 (-) in CD2 Cl2 , however, show a blue-shift compared to the corresponding BPh4 (-) salt.

A comprehensive microscopic picture of the benzhydryl radical and cation photogeneration and interconversion through electron transfer.

Bond cleavage and bond formation are central to organic chemistry. Carbocations play a key role in our understanding of nucleophilic substitution reactions that involve both processes. The precise understanding of the mechanism and dynamics of the photogeneration of carbocations and carbon radicals is therefore an important quest. In particular, the role of electron transfer for the generation of carbocations from the radical pair is still unclear. A quantitative femtosecond absorption study is presented, with ultrabroad probing on selected donor and acceptor substituted benzhydryl chlorides irradiated with 270 nm (35 fs) pulses. The ultrafast bond cleavage within 300 fs is almost exclusively homolytic, thus leading to a radical pair. The carbocations observable in the nanosecond regime are generated from these radicals by electron transfer from the benzhydryl to the chlorine radical within the first tens of picoseconds. Their concentration is reduced by geminate recombination within hundreds of picoseconds. In moderately polar solvents this depletion almost extinguishes the cation population; in highly polar solvents free ions are still observable on the nanosecond timescale. The explanation of the experimental findings requires the microscopic realm of the intermediates to be accounted for, including their spatial and environmental distributions. The distance dependent electron transfer described by Marcus theory is combined with Smoluchowski diffusion. The depletion of the radical pair distribution at small distances causes a temporal increase of the mean distance and the observed stretched exponential electron transfer. A close accord with experiment can only be reached for a broad distribution of the nascent radical pairs. The increase in the inter-radical and inter-ion pair distance is measured directly as a shift of the UV/Vis absorption of the products. The results demonstrate that, at least for aprotic solvents, traditional descriptions of reaction mechanisms based on the concept of contact and solvent-separated pairs have to be reassessed.

Ground and excited state surfaces for the photochemical bond cleavage in phenylmethylphenylphosphonium ions.

Photolytic bond cleavage is a well-established method to generate carbocations for organic synthesis. Changes in the leaving group have a large influence on the chemical yield. The underlying potential energy surfaces governing the initial process are mostly unknown. We provide potential energy surfaces of ground and excited states on the CASSCF/CASPT2 level of theory for the charged precursor phenylmethylphenylphosphonium ion. We present the electronic and structural changes accompanying the excitation process and the subsequent bond cleavage. Inter-ring charge-transfer processes play a crucial role in the Franck-Condon region. Beyond the Franck-Condon region, competing reaction pathways emerge connected through conical intersections. The phenylmethylphenylphosphonium ion is used as a model system for the commonly used diphenylmethyltriphenylphosphonium ion. The appropriateness of the model is tested by CC2 calculations of the excitation spectrum.

Optimal Bond Constraint Topology for Molecular Dynamics Simulations of Cholesterol.

We recently observed artificial temperature gradients in molecular dynamics (MD) simulations of phase-separating ternary lipid mixtures using the Martini 2 force field. We traced this artifact to insufficiently converged bond length constraints with typical time steps and default settings for the linear constraint solver (LINCS). Here, we systematically optimize the constraint scaffold of cholesterol. With massive virtual sites in an equimomental arrangement, we accelerate bond constraint convergence while preserving the original cholesterol force field and dynamics. The optimized model does not induce nonphysical temperature gradients even at relaxed LINCS settings and is at least as fast as the original model at the strict LINCS settings required for proper thermal sampling. We provide a python script to diagnose possible problems with constraint convergence for other molecules and force fields. Equimomental constraint topology optimization can also be used to boost constraint convergence in atomistic MD simulations of molecular systems.

A locally activatable sensor for robust quantification of organellar glutathione.

Glutathione (GSH) is the main determinant of intracellular redox potential and participates in multiple cellular signalling pathways. Achieving a detailed understanding of intracellular GSH homeostasis depends on the development of tools to map GSH compartmentalization and intra-organelle fluctuations. Here we present a GSH-sensing platform for live-cell imaging, termed targetable ratiometric quantitative GSH (TRaQ-G). This chemogenetic sensor possesses a unique reactivity turn-on mechanism, ensuring that the small molecule is only sensitive to GSH in a desired location. Furthermore, TRaQ-G can be fused to a fluorescent protein to give a ratiometric response. Using TRaQ-G fused to a redox-insensitive fluorescent protein, we demonstrate that the nuclear and cytosolic GSH pools are independently regulated during cell proliferation. This sensor was used in combination with a redox-sensitive fluorescent protein to quantify redox potential and GSH concentration simultaneously in the endoplasmic reticulum. Finally, by exchanging the fluorescent protein, we created a near-infrared, targetable and quantitative GSH sensor.

Martini 3 Coarse-Grained Force Field for Cholesterol.

Cholesterol plays a crucial role in biomembranes by regulating various properties, such as fluidity, rigidity, permeability, and organization of lipid bilayers. The latest version of the Martini model, Martini 3, offers significant improvements in interaction balance, molecular packing, and inclusion of new bead types and sizes. However, the release of the new model resulted in the need to reparameterize many core molecules, including cholesterol. Here, we describe the development and validation of a Martini 3 cholesterol model, addressing issues related to its bonded setup, shape, volume, and hydrophobicity. The proposed model mitigates some limitations of its Martini 2 predecessor while maintaining or improving the overall behavior.

Optimal control theory--closing the gap between theory and experiment.

Optimal control theory and optimal control experiments are state-of-the-art tools to control quantum systems. Both methods have been demonstrated successfully for numerous applications in molecular physics, chemistry and biology. Modulated light pulses could be realized, driving these various control processes. Next to the control efficiency, a key issue is the understanding of the control mechanism. An obvious way is to seek support from theory. However, the underlying search strategies in theory and experiment towards the optimal laser field differ. While the optimal control theory operates in the time domain, optimal control experiments optimize the laser fields in the frequency domain. This also implies that both search procedures experience a different bias and follow different pathways on the search landscape. In this perspective we review our recent developments in optimal control theory and their applications. Especially, we focus on approaches, which close the gap between theory and experiment. To this extent we followed two ways. One uses sophisticated optimization algorithms, which enhance the capabilities of optimal control experiments. The other is to extend and modify the optimal control theory formalism in order to mimic the experimental conditions.

Two cooperative binding sites sensitize PI(4,5)P2 recognition by the tubby domain.

Phosphoinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. The dominant PI of the plasma membrane is phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. One of only two PI(4,5)P2 recognition domains characterized in detail is the tubby domain. It is essential for targeting proteins into cilia involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic. Here, we used coarse-grained molecular dynamics simulations to explore PI(4,5)P2 binding by the prototypic tubby domain. The comparatively low PI(4,5)P2 affinity of the previously described canonical binding site is underpinned in a cooperative manner by a previously unknown, adjacent second binding site. Mutations in the previously unknown site impaired PI(4,5)P2-dependent plasma membrane localization in living cells and PI(4,5)P2 interaction in silico, emphasizing its importance for PI(4,5)P2 affinity. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of tubby-like proteins that underlies delivery of ciliary cargo.

Hypothesis-Driven, Structure-Based Design in Photopharmacology: The Case of eDHFR Inhibitors.

Photopharmacology uses light to regulate the biological activity of drugs. This precise control is obtained through the incorporation of molecular photoswitches into bioactive molecules. A major challenge for photopharmacology is the rational design of photoswitchable drugs that show light-induced activation. Computer-aided drug design is an attractive approach toward more effective, targeted design. Herein, we critically evaluated different structure-based approaches for photopharmacology with Escherichia coli dihydrofolate reductase (eDHFR) as a case study. Through the iterative examination of our hypotheses, we progressively tuned the design of azobenzene-based, photoswitchable eDHFR inhibitors in five design-make-switch-test-analyze cycles. Targeting a hydrophobic subpocket of the enzyme and a specific salt bridge only with the thermally metastable cis-isomer emerged as the most promising design strategy. We identified three inhibitors that could be activated upon irradiation and reached potencies in the low-nanomolar range. Above all, this systematic study provided valuable insights for future endeavors toward rational photopharmacology.