Entropy Tug-of-War Determines Solvent Effects in the Liquid-Liquid Phase Separation of a Globular Protein.

Liquid-liquid phase separation (LLPS) plays a key role in the compartmentalization of cells via the formation of biomolecular condensates. Here, we combined atomistic molecular dynamics (MD) simulations and terahertz (THz) spectroscopy to determine the solvent entropy contribution to the formation of condensates of the human eye lens protein γD-Crystallin. The MD simulations reveal an entropy tug-of-war between water molecules that are released from the protein droplets and those that are retained within the condensates, two categories of water molecules that were also assigned spectroscopically. A recently developed THz-calorimetry method enables quantitative comparison of the experimental and computational entropy changes of the released water molecules. The strong correlation mutually validates the two approaches and opens the way to a detailed atomic-level understanding of the different driving forces underlying the LLPS.

A Dynamic Water Channel Affects O2 Stability in [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O2 ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4FeH ) and a unique diiron moiety (2FeH ). Previous attempts to prevent O2 -induced damage have focused on enhancing the protein's sieving effect for O2 by blocking the hydrophobic gas channels that connect the protein surface and the 2FeH . In this study, we aimed to block an O2 diffusion pathway and shield 4FeH instead. Molecular dynamics (MD) simulations identified a novel water channel (WH ) surrounding the H-cluster. As this hydrophilic path may be accessible for O2 molecules we applied site-directed mutagenesis targeting amino acids along WH in proximity to 4FeH to block O2 diffusion. Protein film electrochemistry experiments demonstrate increased O2 stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O2 in the environment of the 4FeH in both cases. The results strongly suggest that, in wild type proteins, O2 diffuses from the 4FeH to the 2FeH . These results reveal new strategies for improving the O2 stability of [FeFe]-hydrogenases by focusing on the O2 diffusion network near the active site.

Structural basis for triacylglyceride extraction from mycobacterial inner membrane by MFS transporter Rv1410.

Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein LprG are involved in transport of triacylglycerides (TAGs) that seal the mycomembrane. Here, we report a 2.7 Å structure of a mycobacterial Rv1410 homologue, which adopts an outward-facing conformation and exhibits unusual transmembrane helix 11 and 12 extensions that protrude ~20 Å into the periplasm. A small, very hydrophobic cavity suitable for lipid transport is constricted by a functionally important ion-lock likely involved in proton coupling. Combining mutational analyses and MD simulations, we propose that TAGs are extracted from the core of the inner membrane into the central cavity via lateral clefts present in the inward-facing conformation. The functional role of the periplasmic helix extensions is to channel the extracted TAG into the lipid binding pocket of LprG.

Dynamics of organophosphate guest encapsulation in heteroleptic coordination cages.

Heteroleptic coordination cages allow the design of different host structures that can bind guest molecules within their cavities. In a previous work, the energetics of organophosphate encapsulation in palladium(II)-based heteroleptic coordination cages that differ in terms of their ability to form hydrogen bonds have been investigated [Platzek et al., Endohedrally Functionalized Heteroleptic Coordination Cages for Phosphate Ester Binding, Angew. Chem., Int. Ed. 2022, 61, e2022093]. The present work focuses on the dynamics of this system. Dynamic information is obtained through the application of a Markov state model (MSM) to unbiased multi-microsecond atomistic molecular dynamics simulations of guest binding and release. The MSM reveals that both the bound state and the binding/unbinding pathways are highly dynamic, with different types of interactions mediating the binding of the diphenylphosphate guest. Thus, the simulations highlight the dynamic nature of the nanoconfinement in the host-guest systems, with possible implications for the use of such coordination cages as catalysts.

Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H+-ATPase.

The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase Arabidopsis thaliana H+-ATPase isoform 2 (AHA2) upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytoplasmic C-terminal regulatory domain of the pump. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partially conserved in functionally different P-type ATPases from different organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.

Viscosity Prediction of High-Concentration Antibody Solutions with Atomistic Simulations.

The computational prediction of the viscosity of dense protein solutions is highly desirable, for example, in the early development phase of high-concentration biopharmaceutical formulations where the material needed for experimental determination is typically limited. Here, we use large-scale atomistic molecular dynamics (MD) simulations with explicit solvation to de novo predict the dynamic viscosities of solutions of a monoclonal IgG1 antibody (mAb) from the pressure fluctuations using a Green-Kubo approach. The viscosities at simulated mAb concentrations of 200 and 250 mg/mL are compared to the experimental values, which we measured with rotational rheometry. The computational viscosity of 24 mPa·s at the mAb concentration of 250 mg/mL matches the experimental value of 23 mPa·s obtained at a concentration of 213 mg/mL, indicating slightly different effective concentrations (or activities) in the MD simulations and in the experiments. This difference is assigned to a slight underestimation of the effective mAb-mAb interactions in the simulations, leading to a too loose dynamic mAb network that governs the viscosity. Taken together, this study demonstrates the feasibility of all-atom MD simulations for predicting the properties of dense mAb solutions and provides detailed microscopic insights into the underlying molecular interactions. At the same time, it also shows that there is room for further improvements and highlights challenges, such as the massive sampling required for computing collective properties of dense biomolecular solutions in the high-viscosity regime with reasonable statistical precision.

Thermodynamic forces from protein and water govern condensate formation of an intrinsically disordered protein domain.

Liquid-liquid phase separation (LLPS) can drive a multitude of cellular processes by compartmentalizing biological cells via the formation of dense liquid biomolecular condensates, which can function as membraneless organelles. Despite its importance, the molecular-level understanding of the underlying thermodynamics of this process remains incomplete. In this study, we use atomistic molecular dynamics simulations of the low complexity domain (LCD) of human fused in sarcoma (FUS) protein to investigate the contributions of water and protein molecules to the free energy changes that govern LLPS. Both protein and water components are found to have comparably sizeable thermodynamic contributions to the formation of FUS condensates. Moreover, we quantify the counteracting effects of water molecules that are released into the bulk upon condensate formation and the waters retained within the protein droplets. Among the various factors considered, solvation entropy and protein interaction enthalpy are identified as the most important contributions, while solvation enthalpy and protein entropy changes are smaller. These results provide detailed molecular insights on the intricate thermodynamic interplay between protein- and solvation-related forces underlying the formation of biomolecular condensates.

Chiroptical Recognition of Carboxylates with Charge-Neutral Double-Stranded Zinc(II) Helicates.

Chirality analysis of small molecules for the determination of their enantiopurity is nowadays ruled by streamlined chromatographic methods which utilize chiral stationary phases. Chiroptical probes which rely on host-guest interactions are so far overshadowed by the latter but have the benefit of depending only on common spectroscopic techniques such as CD spectroscopy to distinguish enantiomers and to quantify their ratio. Interest into this receptor-based approach is constantly rising because non-invasive high-throughput screenings with a minimal waste production can be performed. In this study we investigate the possibility to utilize metal-based containers in form of charge-neutral helicates able to recognize anions for this purpose. Key building block of the helicates are triazole units which show rotational freedom and give rise to either a meso-structure or a racemic mixture of the right- and left-handed complex. A chiroptical response of the probe is observed upon recognition of chiral mono- or dicarboxylates and chirality analysis of tartrate is conducted by CD spectroscopy.

Epigenetic CpG duplex marks probed by an evolved DNA reader via a well-tempered conformational plasticity.

5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel 'CpG duplex marks' could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.

Integrated Assessment of the Structure and Dynamics of Solid Proteins.

Understanding macromolecular function, interactions, and stability hinges on detailed assessment of conformational ensembles. For solid proteins, accurate elucidation of the spatial aspects of dynamics at physiological temperatures is limited by the qualitative character or low abundance of solid-state nuclear magnetic resonance internuclear distance information. Here, we demonstrate access to abundant proton-proton internuclear distances for integrated structural biology and chemistry with unprecedented accuracy. Apart from highest-resolution single-state structures, the exact distances enable molecular dynamics (MD) ensemble simulations orchestrated by a dense network of experimental interproton distance boundaries gathered in the context of their physical lattices. This direct embedding of experimental ensemble distances into MD will provide access to representative, atomic-level spatial details of conformational dynamics in supramolecular assemblies, crystalline and lipid-embedded proteins, and beyond.

Increasing the O2 Resistance of the [FeFe]-Hydrogenase CbA5H through Enhanced Protein Flexibility.

The high turnover rates of [FeFe]-hydrogenases under mild conditions and at low overpotentials provide a natural blueprint for the design of hydrogen catalysts. However, the unique active site (H-cluster) degrades upon contact with oxygen. The [FeFe]-hydrogenase fromClostridium beijerinckii (CbA5H) is characterized by the flexibility of its protein structure, which allows a conserved cysteine to coordinate to the active site under oxidative conditions. Thereby, intrinsic cofactor degradation induced by dioxygen is minimized. However, the protection from O2 is only partial, and the activity of the enzyme decreases upon each exposure to O2. By using site-directed mutagenesis in combination with electrochemistry, ATR-FTIR spectroscopy, and molecular dynamics simulations, we show that the kinetics of the conversion between the oxygen-protected inactive state (cysteine-bound) and the oxygen-sensitive active state can be accelerated by replacing a surface residue that is very distant from the active site. This sole exchange of methionine for a glutamate residue leads to an increased resistance of the hydrogenase to dioxygen. With our study, we aim to understand how local modifications of the protein structure can have a crucial impact on protein dynamics and how they can control the reactivity of inorganic active sites through outer sphere effects.

Synthetic Monosaccharide Channels: Size-Selective Transmembrane Transport of Glucose and Fructose Mediated by Porphyrin Boxes.

Here we report synthetic monosaccharide channels built with shape-persistent organic cages, porphyrin boxes (PBs), that allow facile transmembrane transport of glucose and fructose through their windows. PBs show a much higher transport rate for glucose and fructose over disaccharides such as sucrose, as evidenced by intravesicular enzyme assays and molecular dynamics simulations. The transport rate can be modulated by changing the length of the alkyl chains decorating the cage windows. Insertion of a linear pillar ligand into the cavity of PBs blocks the monosaccharide transport. In vitro cell experiment shows that PBs transport glucose across the living-cell membrane and enhance cell viability when the natural glucose transporter GLUT1 is blocked. Time-dependent live-cell imaging and MTT assays confirm the cyto-compatibility of PBs. The monosaccharide-selective transport ability of PBs is reminiscent of natural glucose transporters (GLUTs), which are crucial for numerous biological functions.

Probing Methyl Group Dynamics in Proteins by NMR Cross-Correlated Dipolar Relaxation and Molecular Dynamics Simulations.

Nuclear magnetic resonance (NMR) spin relaxation is the most informative approach to experimentally probe the internal dynamics of proteins on the picosecond to nanosecond time scale. At the same time, molecular dynamics (MD) simulations of biological macromolecules are steadily improving through better physical models, enhanced sampling methods, and increased computational power, and they provide exquisite information about flexibility and its role in protein stability and molecular interactions. Many examples have shown that MD is now adept in probing protein backbone motion, but improvements are still required toward a quantitative description of the dynamics of side chains, for example, probed by the dynamics of methyl groups. Thus far, the comparison of computation with experiment for side chain dynamics has primarily focused on the relaxation of 13C and 2H nuclei induced by autocorrelated variation of spin interactions. However, the cross-correlation of 13C-1H dipolar interactions in methyl groups offers an attractive alternative. Here, we establish a computational framework to extract cross-correlation relaxation parameters of methyl groups in proteins from all-atom MD simulations. To demonstrate the utility of the approach, cross-correlation relaxation rates of ubiquitin are computed from MD simulations performed with the AMBER99SB*-ILDN and CHARMM36 force fields. Simulation results were found to agree well with those obtained by experiment. Moreover, the data obtained with the two force fields are highly consistent.

Partial peptide dissociation and binding groove plasticity in two major histocompatibility complex class I alleles - differences between alleles versus force field and sampling effects.

Major histocompatibility complex class I (MHC I) reports a cell's health status by presenting antigenic peptides inside its binding groove. However, MHC I binding grooves can differ largely in their plasticity, from binding grooves that are conformationally stable by themselves to those that require a high-affinity peptide to be bound to attain conformational stability. These latter MHC I alleles are dependent on the C-terminus of the peptide that stabilizes the F-pocket region of their binding grooves. It has remained unclear to what extent a peptide-MHC I complex (pMHC I) can tolerate the (intermittent) partial dissociation of high-affinity peptides, especially of the peptide's N-terminus. Using bias exchange umbrella sampling (BEUS), a technique to achieve enhanced sampling in molecular dynamics (MD) simulations, we obtained the free-energy profiles of the N-terminal dissociation of a respective high-affinity peptide from HLA-B*35:01 and HLA-B*44:02, two alleles on opposite ends of the scale regarding binding groove plasticity. The potential of mean force (PMF) for HLA-B*35:01 was calculated for two different sets of starting structures and is compared with a PMF obtained previously with a different force field to disentangle allele differences from force field and sampling effects. For both alleles, the free-energy profiles indicate that the peptide N-terminus dissociates in a substantial fraction of the pMHC I, suggesting that their crystal structures with fully bound peptides only partially capture the dynamic conformational ensemble of pMHC I in solution, and thus in the cell.

The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells.

Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells.

Endohedrally Functionalized Heteroleptic Coordination Cages for Phosphate Ester Binding.

Metallosupramolecular hosts of nanoscopic dimensions, which are able to serve as selective receptors and catalysts, are usually composed of only one type of organic ligand, restricting diversity in terms of cavity shape and functional group decoration. We report a series of heteroleptic [Pd2 A2 B2 ] coordination cages that self-assemble from a library of shape complementary bis-monodentate ligands in a non-statistical fashion. Ligands A feature an inward pointing NH function, able to engage in hydrogen bonding and amenable to being functionalized with amide and alkyl substituents. Ligands B comprise tricyclic aromatic backbones of different shape and electronic situation. The obtained heteroleptic coordination cages were investigated for their ability to bind phosphate diesters as guests. All-atom molecular dynamics (MD) simulations in explicit solvent were conducted to understand the mechanistic relationships behind the experimentally determined guest affinities.

Steering the Ultrafast Opening and Closure Dynamics of a Photochromic Coordination Cage by Guest Molecules.

Photochemical studies on supramolecular hosts that can encapsulate small guest molecules commonly focus on three aspects: photoswitching the cage to release or trap the guest, the effect of the confining environment on the guest, and light-induced exciton or charge transfer within the cage structure. Here, we exploit ultrafast spectroscopy to address how the guest alters the photoswitching characteristics of the cage. For this, the impacts of three disparate guest compounds on ring-opening or ring-closure of a dithienylethene (DTE) ligand in a photoswitchable DTE-based coordination cage are juxtaposed. The guest modulates both outcome and timescale of the cage's photodynamics, by an interplay of structural strain, heavy-atom effect, and enhancement of charge-transfer processes exercised by the guest on the photo-excited cage. The approach might prove beneficial for attuning the applicability of photoswitchable nanocontainers and desired guest compounds.

Spatially Resolved Hydration Thermodynamics in Biomolecular Systems.

Water is essential for the structure, dynamics, energetics, and thus the function of biomolecules. It is a formidable challenge to elicit, in microscopic detail, the role of the solvation-related driving forces of biomolecular processes, such as the enthalpy and entropy contributions to the underlying free-energy landscape. In this Perspective, we discuss recent developments and applications of computational methods that provide a spatially resolved map of hydration thermodynamics in biomolecular systems and thus yield atomic-level insights to guide the interpretation of experimental observations. An emphasis is on the challenge of quantifying the hydration entropy, which requires characterization of both the motions of the biomolecules and of the water molecules in their surrounding.

VCD spectroscopy reveals conformational changes of chiral crown ethers upon complexation of potassium and ammonium cations.

Two chiral derivatives of 18-crown-6, namely the host molecules 2,3-diphenyl- and 2-phenyl-18c6, serve as model systems to investigate whether VCD spectroscopy can be used to monitor conformational changes occurring upon complexation of guests. Host-guest complexes of both crown ethers were prepared by addition of KNO3. The more bulky 2,3-diphenyl-18c6 is found to undergo major conformational changes upon encapsulation of K+, which are revealed as characteristic changes of the VCD spectral signatures. In contrast, while 2-phenyl-18c6 also incorporates K+ into the macrocycle, strong conformational changes are not occurring and thus spectral changes are negligible. With an octyl ammonium cation as guest molecule, 2,3-diphenyl-18c6 shows the same conformational and spectral changes that were observed for K+-complexes. In addition, the asymmetric NH3-deformation modes are found to gain VCD intensity through an induced VCD process. An analysis of the vibrational spectra enables a differentiation of VCD active and inactive guest modes: There appears to be a correlation between the symmetry of the vibrational mode and the induced VCD intensity. While this finding makes the host-guest complexes interesting systems for future theoretical studies on the origin of induced VCD signatures, the observations described in this study demonstrate that VCD spectroscopy is indeed a suitable technique for the characterization of supramolecular host-guest complexes.

Accurate evaluation of combustion enthalpy by ab-intio computations.

Accurate evaluation of combustion enthalpy is of high scientific and industrial importance. Although ab-initio computation of the heat of reactions is one of the promising and well-established approaches in computational chemistry, reliable and precise computation of heat of combustion reactions by ab-initio methods is surprisingly scarce in the literature. A handful of works carried out for this purpose report significant inconsistencies between the computed and experimentally determined combustion enthalpies and suggest empirical corrections to improve the accuracy of the ab-initio predicted data. The main aim of the present study is to investigate the reasons behind those reported inconsistencies and propose guidelines for a high-accuracy estimation of heat of reactions via ab-initio computations. We show comparably accurate prediction of combustion enthalpy of 40 organic molecules based on a DSD-PBEP86 double-hybrid density functional theory approach and CCSD(T)-F12 coupled-cluster computations, with mean unsigned errors with respect to experimental data being below 0.5% for both methods.