Physical Chemistry Chemical Physics

We present a comparative study of the inner membrane of three Gram-positive bacterial strains, namely S. aureus, S. epidermidis and N. lacusekhoensis. A lipidomics study is used to obtain the lipid architecture and composition for S. epidermidis found in the skin microbiome and N. lacusekhoensis, an extremophile present in halophilic and alkophilic environments. Differences between the strains arise from both the lipid architecture and the cardiolipin content varying from 5% in S. aureus to 85% in N. lacusekhoensis. We develop coarse grained (CG) Martini-3 membrane models which reproduce structural properties such as membrane area, thickness, density distributions as well as ion-correlations with all-atom models. Inter-lipid correlations reveal a homogeneous distribution of lipids in the membranes despite the wide variation in lipid types and composition. Mechanical properties such as the area stretch modulus increased with cardiolipin content, however the bending modulus has a more complex dependence on membrane charge and lipid type. Using the CG models we evaluate the insertion free energies for four widely used antimicrobial molecules. Entry barriers for thymol and methylparaben arise from the charge density modulation at the membrane headgroups due to counterion condensation. The entry mechanisms of the antimicrobial peptide cecropin-melittin-15 (CM15) and the preservative molecule ethyl-lauroyl-arginate (ELAR) are found to be similar across all three strains. We also illustrate the manner in which the extremophilic strain, N. lacusekhoensis with its high cardiolipin content, modulates the partitioning kinetics of the antimicrobial molecule thymol with pH and salt. Our study reveals that membrane properties are largely conserved across the three model membranes. The molecular models and insights emerging from the present work should aid in the development of novel antimicrobials against Gram-positive strains.

The process of drug unbinding is of immense importance in the field of biophysics and therapeutics. The behavior of these systems is greatly influenced by their thermodynamic and kinetic properties. Therefore, it is crucial to accurately estimate the ligand binding free energies and rate of ligand dissociation, yet these processes are often governed by rare event transitions that lie beyond the reach of standard brute-force molecular dynamics simulations. While enhanced sampling simulations offer a solution, their efficacy is strictly contingent upon the selection of appropriate collective variables (CVs) which is non-trivial for complex systems like protein-ligand complexes. In this study, we present a method to derive optimized CV from transition state region (TS) via an interpretable machine learning (ML) model, Elastic Net. By employing some physically intuitive order parameters, the derived optimized CV from the TS-region greatly accelerate ligand binding-unbinding transitions and achieves rapid free energy surface (FES) convergence across diverse systems including buried and solvent exposed active sites such as Trpsin-benzamidine complex, host-guest systems and sodium epoxidase etc. Intriguingly significant contribution of the ligand hydration is found in the optimized CV which depicts crucial role of solvent in driving ligand binding-unbinding transitions. The estimated binding free energies for different protein-ligand complexes match quite well with experiments, while maintaining a low computational cost. The derived optimized CV is also used to calculate the ligand residence times across different systems and calculated residence times are within the experimental range for all systems, again with very little computational costs. Moreover, we show that the optimized CV constructed from TS region via an interpretable ML model is transferable across diverse systems, offering a robust and scalable framework for drug discovery and investigation of complex biomolecular recognition.

Amylose contributes to starch crystallinity, but the stability of packed amylose double helices in water at elevated temperature remains insufficiently characterized. Here, we used molecular dynamics simulations to test whether chain length affects the short-timescale stability of A-type amylose oligomers in water. Six systems differing in chain length (6, 12, or 24 glucose units per chain) and oligomer size (isolated double strand or dodecamer of six double strands) were simulated, and five independent 1-s production runs were analyzed for each simulated condition. Oligomers with six glucose units showed structural collapse accompanied by increased water penetration. By contrast, dodecamers with 12 or 24 glucose units largely retained packed double-helical organization over the simulated timescale, although fraying was observed at their ends. These results indicate that chain length and lateral packing strongly affect the early structural response of amylose-like crystalline segments in hot water. The present simulations do not establish the ultimate fate of longer oligomers at longer timescales, but they identify a relative stability difference that is relevant to molecular interpretations of hydration-driven disordering in starch.

Molecular dynamics simulations of nucleic acids are performed using a solvent-buffer distance of 10 [A] between the solute surface and the simulation box boundary. Although this cell size has been extensively explored in protein simulations, its implications for nucleic acid dynamics are not well understood. Nucleic acids are elongated, highly charged, and flexible structures with hydration and dynamical properties distinct from those of proteins and therefore, they may require different solvent-layer considerations in simulations. In this study, we investigated the effect of simulation cell size on nucleic acid dynamics by simulating a 30-base-pair double-helical nucleic acid structure and its two single-stranded forms using solvent-buffer distances of 3, 5, 10, 15, and 20 [A]. Smaller cells may impose restricted hydration, molecular crowding, and periodic image interactions. However, larger cells provide solvent space for conformational relaxation. A total of 45 s of molecular dynamics simulations were performed (3 structures x 5 cell sizes x 3 replicates x 1 s). Our results show that while the commonly used 10 [A] buffer may be sufficient to maintain the stability of the double-stranded nucleic acid, larger cells are required to capture the conformational dynamics of single-stranded structures. In both, increasing the cell size to 15 or 20 [A] enables broader conformational sampling. The first hydration shell exhibits reduced crowding in the 20 [A] cell, consistent with more relaxed conformations. At larger cell sizes, single-stranded nucleic acids adopt compact, self-associated conformations for stability. Together, this study presents physical insight into how simulation cell size and solvent environment influence nucleic acid dynamics.

The High Mobility Group Box 1 (HMGB1) protein performs multiple essential functions in the body, ranging from DNA regulation to the activation and mediation of immune responses. However, HMGB1 has been also implicated in several pathological conditions, such as rheumatoid arthritis, sepsis, autoimmune diseases, tumors, and Alzheimer's disease. As a result, HMGB1 is of increasing interest as a therapeutic target. Binding to heparin has been reported to inhibit HMGB1's pathological activity during sepsis in clinical settings. In this work, we compare the interactions of HMGB1 with heparin and its' synthetic analog linear polyglycerol sulfate (lPGS) from the viewpoint of stability and changes to association behavior. This analysis focuses on thermal stability, secondary-structure changes, and particle-size evolution using nano-differential scanning fluorimetry (nanoDSF), circular dichroism spectroscopy (CD), and dynamic light scattering (DLS).

Far-Red (FR) Light Photoacclimation (FaRLiP) enables cyanobacteria to extend photosynthetic activity into the far-red region by extensively remodelling Photosystem I (PSI), including the replacement of several core subunits with paralogs that coordinate the red-shifted chlorophyll f (Chl f). The binding positions of Chls f are still a matter of debate, with the most recent structural findings supporting the location of a single Chl f molecule within the reaction centre (RC) at the so-called A-1B site. This was in turn suggested to strongly affect electron transfer (ET) directionality leading to an almost monodirectional transfer along the B branch in FR-PSI RC. Here, we directly probe ET in FR-PSI by characterising the photogenerated [P700A1-] spin-correlated radical pair using complementary pulse and Time-Resolved (TR) Electron Paramagnetic Resonance (EPR) spectroscopy at cryogenic temperature. Electron spin-echo decay kinetics are distinctly biexponential, indicating the formation of two charge-separated states. Consistently, out-of-phase ESEEM traces are quantitatively described by two modulation frequencies arising from different dipolar interactions, while TR-EPR spectra are accurately simulated by the combined contributions of [P700A1A-] and [P700A1B-] radical pairs. These results provide direct spectroscopic evidence that both the A and B branches remain photochemically active in FR-PSI. The conservation of bidirectional ET, even when considering the presence of a single Chl f molecule in the RC, further implies that the two radical pairs originate from a common primary electron donor. This finding identifies P700 as the most likely primary donor and argues against a mechanism in which the RC Chl f initiates charge separation.

The methylation of cytosines at the 5th position (d5mC) is one of the most common epigenetic modifications, and alterations in methylation profile of cells are known to be involved in progression of many diseases including cancer. Increased stability of DNA accompanied by decreased flexibility upon methylation is thought to be a reason behind methylation profiles. The effects of d5mC on DNA stability and structure were investigated via systematic changes in the number and position of d5mCs in DDD. Our results revealed that d5mC substitutions changed DNA conformation and increased the stability only slightly. Next, the effect of DNA methylation on DNA-small molecule interactions was investigated using DDD and fully methylated analogue, DDD8. All the molecules examined (EtBr, Dox, Net and Hoe) had slightly higher affinity to DDD8 compared to DDD. Conversely, their effect, especially Doxs, on DDD structure was more pronounced. Further investigations via MD simulations revealed high selectivity of Dox towards a single intercalation site where the methoxy group of Dox interacts with the methyl group of d5mC and that of two precedent dT to create a highly stable hydrophobic cluster. Hydrophobic cluster formation was not observed upon Dox binding to DDD. Our results rationalize the increased stability of DDD8 over DDD, and open new routes for the design of drugs targeting epigenetic modifications. We suggest, the design of drugs that can engage in hydrophobic interactions with methyl groups in the major groove of a 5-dTdTd5mCdG-3 sequence might lead the way in specific targeting of hypermethylated regions in cancer cells.

1.Surface functionality and pH play a decisive role in governing the structural dynamics, hydration, and drug-binding behaviour of dendrimers. Here, all-atom molecular dynamics (MD) simulations were performed on five generations of PAMAM (G1-G5) and PETIM (G2-G6) dendrimers with O-core and N-core architectures, functionalized with amine, carboxylic acid, or sugar terminal groups under different protonation states. Protonation of the tertiary branch-point amines expands the dendrimer structure, increases internal porosity and hydration, and enhances structural fluctuations across both families. In contrast, non-protonated amine -NH2 (NP) and carboxylic acid -COOH (NP) terminated dendrimers, together with deprotonated carboxylate-COO- (DeP) systems, retain comparatively compact conformations. Sugar-functionalized dendrimers ({beta}-galactose-terminated PETIM and D-glucose-terminated PAMAM) are most hydrated and structurally rigid, whereas amine-terminated dendrimers exhibit the greatest conformational dynamics. PAMAM dendrimers with -NH2, -NH3+, and -COO- terminal groups are generally more hydrated than their PETIM counterparts. However, {beta}-galactose-terminated PETIM dendrimers are more hydrophilic than D-glucose-terminated PAMAM dendrimers. N-core PETIM dendrimers also adopt more compact and spherical conformations than equivalent O-core PETIM dendrimers. Drug-binding MD simulations show that curcumin binding is dominated by van der Waals (vdW) interactions, whereas doxorubicin complexation is primarily driven by electrostatic interactions. Among the investigated surface functionalities, -NH2 (NP), -NH3+ (P), -COOH (NP), and -COO- (DeP) terminations exhibit the most favourable drug-binding characteristics. Except for deprotonated carboxylate systems, curcumin binds more strongly than doxorubicin. Overall, these findings establish molecular-level relationships between surface functionality, protonation state, dendrimer architecture, and drug-binding behaviour, providing design principles for pH-responsive dendrimer nanocarriers with enhanced drug-loading and controlled-release performance. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/742721v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@119bf29org.highwire.dtl.DTLVardef@1554d86org.highwire.dtl.DTLVardef@154a254org.highwire.dtl.DTLVardef@16d5c5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Live cell plasma membranes show spatially heterogeneous liquid-ordered (Lo)-like and liquid-disordered (Ld)-like regions similar to the co-existing Lo/Ld phases observed in lipid vesicles. The Lo-like regions are relatively less hydrated and less polar due to tight packing of the membrane components compared to the Ld-like regions. The steady-state fluorescence spectra of Di-4-ANEPPDHQ (Di-4), a widely used polarity-sensitive probe, is blue or red shifted when solvated in less polar (Ld- like) or more polar (Lo-like) regions respectively. However, quantification of Di-4 fluorescence in blue and red channels for the evaluation of membrane phase state suffers from the lack of specific wavelength choice for these two channels and Di-4s relatively higher concentration in Ld phase (red channel) due to its partitioning preference. To address these issues, we employed fluorescence lifetime of Di-4, a concentration independent photophysical parameter, to understand membrane biophysical properties. The fluorescence lifetime of Di-4 in lipid vesicles exhibits Arrhenius-like temperature dependence. Centred around this energetic feature of Di-4 photophysics, we developed a novel analytical module, namely excited state relaxation activation energy (ESRAct), that serves as an intrinsic descriptor of the membrane nano-environment sensed by this probe. We show that the ESRAact value scales with increasing disorder in nanoscale phase separation (i.e., ESRAct of pure Ld > mixed Ld/Lo > pure Lo phase). We then extended its applications to giant plasma membrane vesicles (GPMVs) isolated from MCF-7 cells and found that these vesicles exhibit nanoscale Lo/Ld co-existing phase within 16-37{degrees}C. We envisage wide applications of ESRAct to delineate plasma membrane phase behavior as well as general photophysical studies on other newly designed polarity-sensitive probes.

Photoprotection against excess energy is essential for the survival of photosynthetic organisms under adverse conditions. In plants, excess energy can be dissipated as heat through non-photochemical quenching (NPQ) of chlorophyll fluorescence, involving the trimeric light-harvesting complex II (LHCII), the major antenna of photosystem II. How NPQ affects antenna proteins remains debated, especially as most studies focus on short-lived components artificially induced in vitro. Here, we characterize the effects of qH, a long-lived NPQ component, on the fluorescence properties of natively quenched LHCII. Single-molecule fluorescence measurements, combined with biochemical and biophysical ensemble approaches, reveal a larger and more quenched subpopulation of LHCII trimers exhibiting fluorescence intermittency in samples with qH compared to those without. This behavior is linked to a small conformational change that stabilizes a quenched state, enhancing photoprotection at the antenna level. These findings provide new insights into sustained NPQ and its role in regulating energy dissipation under natural light conditions.

Keratin proteins are fundamental structural components of hair fibers, contributing to their mechanical resilience, elasticity, and fracture resistance. However, systematic molecular-scale characterization of keratin unfolding mechanics across protein types remains limited, restricting the connection between protein-level deformation mechanisms and hierarchical hair fiber mechanics. Here, we establish a comparative molecular-dynamics-based framework for characterizing the unfolding behavior and nanomechanical response of a curated dataset of 51 keratin proteins. We conduct implicit atomistic molecular dynamics (MD) simulations, including equilibration and steered molecular dynamics (SMD) under four accelerated pulling velocities, to quantify unfolding forces, energy absorption, and structure-property relationships. These accelerated pulling conditions are interpreted as computational probes of relative molecular-scale trends, rather than direct reproductions of experimental hair-fiber strain-rate regimes. Across these accelerated SMD conditions, the simulations show rate-sensitive increases in unfolding force and energy absorption, consistent with constrained molecular relaxation during faster molecular pulling. Stronger correlations between nanomechanical properties and molecular descriptors emerge at higher pulling rates, and the nanomechanical responses of different keratin types (Type I and II) are also compared. The findings provide molecular-level insights into protein unfolding mechanisms that may contribute to the mechanical behavior of hierarchical keratin structures. This study establishes a quantitative framework for comparative keratin unfolding mechanics, providing molecular-level descriptors for future multiscale modeling of hair fiber behavior. These results support applications in biomaterial design, hair fiber durability analysis, and bioinspired material engineering. Future work will integrate these nanomechanical descriptors with fiber-level mechanics and machine learning-based keratin design.

The mechanical response of the interaction between the T cell receptor (TCR) and the peptide major histocompatibility complex (pMHC) is fundamental to antigen recognition, but the atomic-scale mechanisms by which the CD8 coreceptor modulates the complexs conformational states and force-bearing behavior remain poorly understood. We employed all-atom molecular dynamics and steered molecular dynamics simulations of membrane-embedded TCR-pMHC and TCR-pMHC-CD8 complexes to characterize their dynamics and force-induced dissociation. Microsecond-long molecular dynamics (MD) simulations show that binding of CD8 to the MHC 3 domain applies restraints to the latter one, which leads the MHC 1 helix to stably bind against the TCR complementarity-determining regions (CDRs) and suppresses the fluctuations of the antigenic peptide. Furthermore, under mechanical loading, the TCR-pMHC-CD8 system exhibits a distinct dissociation pathway compared to that of TCR-pMHC complex, which may strengthen the mechanical stability of the binding of TCR-pMHC. Collectively, these findings unravel the molecular mechanisms of CD8-mediated synergistic stabilization and mechanical regulation of TCR-pMHC, providing new mechanistic insights into coreceptor-dependent T cell antigen recognition. SIGNIFICANCEThe TCR-pMHC handshake is the definitive spark that ignites the immune response. It has been shown that the force applied to the TCR-pMHC complex is critical for triggering the downstream signaling. In addition, TCR recognition of pMHC is regulated by force and strongly influenced by coreceptors, such as CD8. Therefore, understanding how coreceptors mediate TCR-pMHC interactions upon the application of force is crucial for revealing the biophysical basis of immune signaling. This study unravels how coreceptors shape the dynamics and mechanical response of the TCR-pMHC complex at the atomic level.

Magnetic treatment in tomato seeds (Solanum lycopersicum L.) has been studied as a biotechnological technique to induce a reduction in germination times and enhance plant development. However, the modified cellular mechanisms involved in the reduction of germination times or the improvement of development parameters are not yet clearly established. To explore a possible altered cellular mechanism, the effect of homogeneous static magnetic fields on the structure of the cyclic nucleotide-gated channel 6 (CNGC6), the modification in the organization of POPC lipids in the plasma membrane, and changes in calcium ion mobility were evaluated. For this purpose, coarse-grained molecular dynamics simulations were performed using the Martini 3 model in GROMACS, applying five different magnetic flux densities (0.000, 0.001, 0.010, 0.100, 1.000, and 10.000) T over 1 000 ns. The results showed an anisotropic effect in the longitudinal direction of the protein, which generated heterogeneous behavior among the chains of the homotetramer; this altered the conformation of the CNGC6 channel and modified the pore bottleneck. In contrast, no significant changes were observed in the conformational order of the POPC phospholipid chains. As a preliminary, single-replicate exploratory study, these results suggest that homogeneous static magnetic fields may induce specific structural modifications in the CNGC6 ion channel of Solanum lycopersicum L. without compromising the integrity of the lipid bilayer or the dynamics of ion transport within the analyzed timescale; these preliminary findings provide a molecular-level structural basis for future experimental and computational investigations of magnetic field effects on plant cyclic nucleotide-gated channels.

Nitric oxide (NO) is a key signaling molecule that regulates diverse physiological responses, including adaptation to hypoxia in plants. Although stabilization of group-VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factors under low O2 is known to be facilitated by the N-dragon pathway, whether NO directly mediates N-terminal cysteine (Cys) oxidation to regulate ERFVII stability remains unresolved. Here, we combined genetic, computational, and structural approaches to investigate NOs role in proteasomal degradation of ERFVII during flooding stress. Arabidopsis mutants with elevated S-Nitrosoglutathione (GSNO) levels compromised ERFVII activation during dark submergence but increased expression of genes encoding N-degron pathway enzymes. Although direct detection of in vivo NO-mediated S-nitrosylated proteins remains technically challenging, the GPS-SNO 1.0 tool predicted the conserved N-terminal second Cys residue as a high-confidence S-nitrosylation site. Structural modeling using the Schrodinger Suite 2024-4 further revealed that conversion of the Cys thiol (Cys-SH) to S-nitrosothiol (Cys-SNO) induced notable conformational changes in ERFVII TFs. Molecular docking further demonstrated that Cys-SNO-modified ERFVII peptides exhibited stronger binding affinities and altered interaction networks with N-degron pathway enzymes, supporting a role for NO-mediated structural remodeling in ERFVII degradation. Elevated GSNO also disrupted energy balance efficiency for overcoming O2 deficiency, altered the expression of sugar starvation-responsive genes, and impaired ATP binding capacity of Cys-SNO ERFVII proteins, as evidenced by docking and molecular dynamics simulations. Collectively, these results support a model in which NO-mediated modification of the conserved N-terminal Cys promotes ERFVII degradation, thereby linking NO signaling to hypoxia-responsive transcriptional regulation and metabolic adaptation during flooding stress.

The aggregation of Tau protein into straight filaments (SFs) and paired helical filaments (PHFs) is central to Alzheimers disease (AD) pathology and a key target for therapeutic inhibition. Graphene quantum dots (GQDs) are biocompatible nanomaterials that have shown promise in inhibiting amyloidogenic protein aggregation across related neurological pathologies. The effect of GQD functionalization on interactions with Tau aggregates (TAs) is poorly understood, though recent evidence suggests that anionic GQDs are effective TA inhibitors. In this study, we survey how GQD functionalization influences binding to SFs and PHFs to guide future development of therapeutic GQDs. We identify binding sites in SFs and PHFs, dock our GQD library to these sites, and perform molecular dynamics simulations on promising complexes, totaling 28 {micro}s of sampling. We discover that anionic GQDs preferentially bind to the positively charged SF large protofilament interface, whereas in PHFs, anionic GQDs have a modest binding preference for the C-shaped curve region. Binding of GQDs at the C-shaped curve in both TAs induces distinct protofilament conformational dynamics resembling a pinching motion to capture the GQD. Together, these binding modes may represent early intermediates of the TA disaggregation mechanism. We find that functional groups capable of possessing a negative charge (e.g., COO-, O-, and S-) produce impressive binding affinities. We propose that enriching these functionalizations during GQD synthesis and preparation, particularly sulfur as it is less studied, may yield more potent TA inhibitors and generalize to other amyloid pathologies with positively charged fibril cores. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/741532v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@78f560org.highwire.dtl.DTLVardef@135834eorg.highwire.dtl.DTLVardef@3f9025org.highwire.dtl.DTLVardef@110ade3_HPS_FORMAT_FIGEXP M_FIG C_FIG

This present work shows that E-hook fragments possess functional structure differences governed by electrostatic interactions and sequence composition. The acidic C-terminal tails of tubulin, known as E-hooks, play a central role in regulating interactions between microtubules and motor proteins, microtubule-associated proteins, and enzymatic modifiers. Despite their functional importance, the intrinsic structural properties of these peptide segments remain poorly characterized due to their intrinsically disordered nature. In this work, we present quantum-mechanically optimized structures of hexamer peptides derived from {beta}-tubulin E-hook sequences. Density functional theory calculations were used to optimize peptide geometries using progressively larger basis sets. From the optimized geometries we calculated theoretical Raman spectra, Ramachandran backbone dihedral distributions, and measured radii of gyration to resolve composition dependent structural tendencies. The combined Raman and conformational analyses provide a systematic computational approach for comparing simulated and experimental Raman spectra of tubulin E-hooks and other intrinsically disordered proteins and offer insight into how E-hooks contribute to the recognition mechanisms underlying the tubulin code.

MHPC512 is a massively parallel, special-purpose supercomputer designed primarily for atomic-level molecular dynamics (MD) simulations of biomolecular systems. It comprises 512 processor units interconnected by a high-speed three-dimensional torus network and employs a custom chip architecture that uses 35-bit fixed-point arithmetic to accelerate computation while controlling precision loss within an acceptable margin. The preprocessor is compatible with GROMACS and AMBER input formats and supports widely used biomolecular force fields (including CHARMM, AMBER, and OPLS/AA), the Neutral Territory method for short-range nonbonded interactions, the k-space Gaussian Split Ewald method for long-range electrostatics, and multiple thermostats, barostats, and integrators. We present a three-tier validation protocol--comparing static energy and virial components, examining ensemble distributions (NVE, NVT, NPT), and evaluating long-time statistical properties--demonstrating that MHPC512 reproduces results consistent with GROMACS and AmberTools. Application examples, including bulk water, dipeptide conformational sampling, folding of fast-folding peptides, membrane-protein systems, lipid self-assembly, and GPCR conformational transitions, further confirm its reliability. MHPC512 has been deployed at multiple supercomputing centers and is publicly accessible, representing a significant advance in high-throughput, large-scale biomolecular MD simulations.

The rising incidence of invasive fungal diseases emphasizes the need for novel therapeutic strategies, including membrane-targeting antifungal agents, which require representative lipid models for detailed molecular-level studies. In this work, we propose a consensus quinary fungal plasma membrane model based on lipidomic literature data, specifically PC:PE:PI:PA:PS phospholipid model with ratio of 44:29:13:8:6. Using a bottom-up approach, we characterized the biophysical properties of this system - with particular emphasis on mechanical parameters such as bending rigidity and area compressibility - by combining molecular dynamics simulations with experimental flicker-noise and ATR-FTIR spectroscopies. Furthermore, we investigated the effect of two key non-phospholipid components: ergosterol and triacylglycerols. Biophysical analysis revealed that DPPI and its specific interactions with DSPS induced the most substantial deviations in baseline membrane parameters, particularly area per lipid, membrane thickness, and area compressibility, while DSPS influenced bending rigidity change and DLiPA primarily affected lipid packing defects. In addition, ergosterol and TGs were found to influence all of the investigated parameters to different degree. Notably, the overall biophysical profile of the proposed FPMM closely mimicked that of natural vesicles derived from yeast lipid extracts, establishing this model may provide a reliable platform for studying fungal membrane biophysics and lipid-targeting interactions.

Neutron scattering is a powerful technique to investigate atomic structures and molecular dynamics of proteins at the nano-scale. When it comes to dynamics, incoherent and coherent scattering respectively provide information on the single and collective dynamics of nuclei. In proteins, hydrogen has the highest incoherent cross-section, and it is common practice to overlook the contribution of coherent terms stemming from all nuclei. However, the fast collective dynamics of heavier nuclei could also be studied if coherent scattering and incoherent scattering were experimentally separated. The recent advent of polarized neutron spectroscopy with sufficient flux and energy resolution has made it possible, and opens new perspectives to investigate the relative importance of coherent scattering and the information it provides on biological samples. The present study reports on the use of polarized quasi-elastic neutron scattering (QENS) and the application of a minimalistic model adapted to both individual and collective dynamics. Using a perdeuterated green fluorescent protein as a model globular protein, the study provides an interpretation of the dynamical parameters obtained with QENS, and a comparative study of the Elastic Coherent and Incoherent Scattering Factor. Based on both experiments and calculations, we discuss the relative importance of distinct and self components of coherent scattering, which is often wrongly assumed to be representative of collective dynamics only. The results highlight the current impediments rendering complicated a straightforward analysis of fast collective dynamics in hydrated protein samples.

Holliday junctions (HJs) are key intermediates of homologous recombination and fundamental building blocks in DNA nanotechnology. Although the canonical antiparallel stacked-X conformation has been extensively characterized by X-ray crystallography, whether parallel HJ conformations exist in aqueous solution remains unresolved. Here, we address this question using extensive atomistic molecular dynamics (MD) simulations and replica-exchange umbrella sampling (REUS) free-energy calculations. Starting from canonical antiparallel HJs, standard MD simulations occasionally revealed spontaneous transitions to parallel conformations on the microsecond timescale without disrupting the DNA duplexes or passing through an open junction intermediate. REUS free-energy profiles confirmed the antiparallel state as the global minimum but also identified the parallel conformation as a well-defined local minimum, indicating it is thermodynamically metastable despite an estimated solution population below 1%. The combination of low equilibrium occupancy, microsecond interconversion dynamics, and the surprisingly close structural similarity between antiparallel and parallel junctions provides a plausible explanation for the lack of direct experimental detection. We further found that the free-energy landscape is only weakly affected by branching-point sequence and salt concentration. These results reconcile the apparent absence of experimental evidence for parallel HJs with their structural feasibility in solution and offer a fresh perspective on the historical debate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/737670v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@bb61a0org.highwire.dtl.DTLVardef@673a7org.highwire.dtl.DTLVardef@192f5adorg.highwire.dtl.DTLVardef@13f4941_HPS_FORMAT_FIGEXP M_FIG C_FIG