The Journal of Physical Chemistry B

Phosphatidylinositol (PI) lipids play a crucial role as a vital lipid component in cell membrane domain formation, contributing to cell signaling. In this study, we investigate the impact of PI lipids on the conformational dynamics of tubulin-associated unit (tau) fibrils through multiscale modelling. While prior experimental work by the Lecomte group has demonstrated the influence of PI lipids on the morphology and secondary structure of tau fragments, a detailed molecular understanding of the binding mechanism between tau and PI-incorporated lipids remains absent. Our molecular dynamics (MD) simulations reveal the intricate molecular mechanisms governing tau binding to PI-incorporated bilayers. Specifically, we conduct MD simulations on lipid patches containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (PG), enabling us to explore conformational changes in the R3-R4 section of tau fibrils. Control simulations are conducted on pure lipid patches without tau fibrils, as well as on tau fibrils within bulk water. Our findings demonstrate that PI-incorporated lipids exhibit a stronger affinity for binding to tau fibrils compared to pure PC/PG lipids. All-atom simulations highlight the potential docking sites for PI headgroups at positively charged residues (Lysine, Arginine) on the tau surface. Moreover, the aggregation of PI lipids facilitates tau binding to the membrane. These results not only enhance our comprehension of the disruption of PI-incorporated bilayers, but also shed light on the stability of the tau over the PI containing bilayers.

Human islet amyloid polypeptide (hIAPP or human amylin) is known to aggregate into amyloid fibrils and is implicated in the development of type II diabetes. Prefibrillar species in particular have been linked to cell loss, prompting detailed investigation of early-stage hIAPP aggregation. Insights into the mechanisms underlying early-stage aggregation and the key intermediate structures formed during aggregation are valuable in understanding disease onset at the molecular level and guiding design of effective therapeutic strategies. Here, we use atomistic molecular dynamics simulations with the finite temperature string method to identify and compare multiple pathways for hIAPP trimer formation in water. We focus on the comparison between trimerization from three disordered hIAPP chains (which we call "3-chain assembly") and trimerization from an hIAPP dimer approached by a single disordered chain (called "2+1 assembly"). We show that trimerization is a process uphill in free energy, regardless of the trimerization mechanism, and that a high free energy barrier of 40 kBT must be crossed in 2+1 assembly compared to a moderate barrier of 12 kBT for 3-chain assembly. We find this discrepancy to originate from differences in molecular-level water interactions involved in the two trimerization scenarios. Furthermore, we find that the more thermodynamically favorable 3-chain assembly begins from a previously identified dimer intermediate exhibiting transient {beta}-sheet character, which is then incorporated into a similar trimer intermediate, suggesting stepwise aggregation dynamics.

Developing molecular models to capture the complex physicochemical architecture of the bacterial cell wall and to study the interaction with antibacterial molecules is an important aspect of assessing and developing novel antimicrobial molecules. We carried out molecular dynamics simulations using an atomistic model of peptidoglycan (PGN) to represent the architecture for Gram-positive Staphylococcus aureus. The model is developed to capture various structural features of the staphylococcal cell wall, such as the peptide orientation, area per disaccharide, glycan length distribution, crosslinking, and pore size. A comparison of the cell wall density and electrostatic potentials is made with a previously developed cell wall model of Gram-negative bacteria, Escherichia coli, and properties for both a single and multilayered structures of the Staphylococcal cell wall are studied. We investigated the interactions of the antimicrobial peptide melittin with the PGN structures. The depth of melittin binding to PGN is more pronounced in E. coli than S. aureus, and consequently the melittin has greater contacts with glycan units of E. coli. Contacts of melittin with the amino acids of peptidoglycan are comparable across both the strains, and the D-Ala residues, which are sites for transpeptidation, show enhanced interactions with melittin. A low energetic barrier is observed for translocation thymol with the four-layered peptidoglycan model. The molecular model developed for Gram-positive PGN allows us to compare and contrast the cell wall penetrating properties with Gram-negative strains and assess for the first time binding and translocation of antimicrobial molecules for Gram-positive cell walls.

The preferred aggregation number of dodeclyphoshocholine (DPC) micelles [Formula] encapsulating dimeric and higher order protein assemblies is difficult to determine via experimental techniques due to uncertainty in dimer geometry and heterogeneity in the conformational ensemble. Dimerization of the Amyloid Precursor Protein transmembrane domain (C99) is a particular step of importance in the production of amyloid-{beta} protein and the amyloid cascade. Molecular dynamics simulations of the C99 dimer and other transmembrane proteins have been performed to compliment micelle-phase protein structure studies. It has often been assumed that the value of [Formula] is the same as that of the pure, empty micelle. Here, we provide a convenient method for testing that assumption, while also accounting for the finite-size effects inherent in computer simulations of micelle self-assembly. Employing large, unbiased, coarsegrained molecular dynamics simulations of DPC and C99 dimer self-assembly, we determined the radius of gyration to be 21.6 {+/-} 2.0 [A] for the micelle-encapsulated dimer, and 16.0 {+/-} 1.0 [A] for the pure DPC micelle. Using these radii of gyration, we performed all-atom simulations of DPC-encapsulated C99 dimers with preferred aggregation numbers of 100 and 54 DPC to test the effect of using an expected versus a naive estimate of aggregation number on the structure of the transmembrane protein dimer. Through atomistic simulations, we determined that the transmembrane dimeric structure displays different characteristics depending on the aggregation number of the micelle, in addition to increased water penetration and micelle defects when the aggregation number is too small.

The extracellular lipid matrix in the stratum corneum (SC) is crucial for generating a skin barrier (permeability) function. The lipid matrix contains three major components; ceramides, cholesterol, and free fatty acids. The broad diversity of ceramides depends on their molecular structures (e.g., hydroxylations and chain lengths) and plays a critical role in maintaining the structural integrity of the lipid matrix. Although recent studies identified a new subclass of ceramide, 1-O-acylceramide NP (CerENP), its precise role in the lipid matrix of SC is still elusive. Herein, we investigate the role of CerENP on the structure and permeability of the SC by molecular dynamics simulations. Our results suggest that the CerENP molecules induce a denser lipid matrix in the lateral dimension in the long periodicity phase model with a bilayer-slab- bilayer structure. Moreover, ethanol permeability analysis indicates that CerENP can suppress molecular permeability through the lipid matrix. This study provides insight into the role of a new subclass of ceramide in the SC, which can lead to our better understanding of skin organization and disease-related barrier dysfunction.

As a consequence of the finite size of globular proteins, it is expected that there should be dispersions in the global melting temperature (Tm) and the denaturation midpoint (Cm). Thermodynamic considerations dictate that the dispersions, {Delta}Tm in Tm and {Delta}Cm in Cm, should decrease with N, the number of residues in the protein. We performed coarse-grained simulations of the Self-Organized Polymer (SOP) model of the multi-domain protein, Adenylate Kinase (ADK) with N = 214, in order to calculate thermal and denaturation unfolding titration curves. The results show that [Formula] and [Formula] are non-zero and follow the previously established (Phys. Rev. Lett. 93 268107 (2004)) thermodynamic [Formula] scaling for proteins accurately. For ADK, the dispersions are small ({approx} 0.004), which implies that the melting temperature is more or less unique, which is unlike in BBL (N =40) where [Formula].

The cationic porphyrin, TMPyP4, is a well-established DNA G-quadruplex (G4) binding ligand that can stabilize different topologies via multiple binding modes. However, TMPyP4 has completely opposite destabilizing and unwinding effect on RNA G4 structures. The structural mechanisms that mediate RNA G4 unfolding remains unknown. Here, we report on the TMPyP4-induced RNA G4 unfolding mechanism studied by well-tempered metadynamics (WT-MetaD) with supporting biophysical experiments. The simulations predict a two-state mechanism of TMPyP4 interaction via a groove-bound and a top-face bound conformation. The dynamics of TMPyP4 stacking on the top tetrad disrupts Hoogsteen H-bonds between guanine bases resulting in the consecutive TMPyP4 intercalation from top-to-bottom G-tetrads. The results reveal a striking correlation between computational and experimental approaches and validate WT-MetaD simulations as a powerful tool for studying RNA G4-ligand interactions.

The dynamics of the aggregated light-harvesting complex (LHCII) associated with its antennae pigments can be crucial for a transition between light harvesting and dissipative states pivotal for non-photochemical quenching (NPQ). To this end, aggregation of chlorophyll-a (CLA) without the LHCII and pigment binding LHCII monomers in the plant thylakoid membranes have been investigated using coarse-grained molecular dynamics simulations at 293 K. Both CLA without the LHCII and pigment-binding LHCII monomers dynamically form and break dimers and higher-order aggregates in thylakoids within the simulation time. The contact lifetime and waiting time distributions of CLA dimers exhibit multiple time scales including most populated fast time scales and less populated slow time scales. The survival probability of CLA dimer in the absence of the LHCII follows a non-exponential decay with multiple residence time scales, leading to a time-dependent rate, unlike conventional rate theory. Such non-exponential decay of survival manifests the emergence of dynamic disorder in CLA without the LHCII resulting from the coupling between time scales of dimer formation and higher-order aggregates. The conformational fluctuations of the LHCII known for inter-CLA coupling variation occur on multiple time scales comparable to the LHCII dimer residence time scales leading to less probable but comparable and more probable slower inter-CLA fluctuations. This indicates the dynamic coupling in the LHCII conformations and their aggregates with the antennae pigments can result in dynamic disorder which will be highly relevant for the light-harvesting efficiency and regulation of NPQ. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/638782v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@5fd895org.highwire.dtl.DTLVardef@8456cforg.highwire.dtl.DTLVardef@5f5c69org.highwire.dtl.DTLVardef@abf6d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

While Alzheimers disease is correlated with the presence of A{beta} fibrils in patient brains, the more likely agents are their precursors, soluble oligomers that may form pores or otherwise distort cell membranes. Using all-atom molecular dynamics simulation we study how presence of fatty acids such as lauric acid changes the stability of pore-forming oligomers built from three-stranded A{beta}42 chains. Such a change would alter the distribution of amyloids in the fatty-acid rich brain environment, and therefore could explain the lower polymorphism observed in A{beta}-fibrils derived from brains of patients with Alzheimers disease. We find that lauric acid stabilizes both ring-like and barrel-shaped models, with the effect being stronger for barrel-like models than for ring-like oligomers.

Aberrant misfolding and progressive aggregation of the intrinsically disordered protein (IDP), -synuclein, are associated with the etiology of several neurodegenerative diseases. However, the structurally heterogeneous ensemble of this IDP and lack of a well-defined binding pocket make it difficult to probe the druggability of -synuclein. Here, by building a comprehensive statistical model of the fuzzy ensemble of a millisecond-long atomistic simulation trajectory of monomeric -synuclein interacting with the small-molecule drug fasudil, we identify exhaustive sets of metastable binding-competent states of -synuclein. The model reveals that the interaction with the drug primes this IDP to explore both more compact and more extended conformational sub-ensemble than those in neat water, thereby broadening its structural repertoire in presence of small-molecule via an entropy expansion mechanism. Subsequent simulation of the dimerisation process shows that similar motif of entropic-expansion mechanism helps fasudil to retard the self-aggregation propensity of -synuclein via trapping it into multiple distinct states of diverse compaction featuring aggregation-resistant long-range interactions. Furthermore, small-molecule binding interactions in dimerisation-competent relatively extended states have a screening effect that hinders the formation of stable dimer contacts. Together, the investigation demonstrates the ability of small-molecules to have an ensemble-modulatory effect on IDPs that can be effectively utilised in therapeutic strategies probing aggregation-related diseases.

Fold-switching metamorphic protein sequences defy the classical "one sequence - one fold" paradigm. The ability of metamorphic proteins to reversibly switch between distinct folds make them attractive de novo protein engineering candidates since they can function as environment-sensitive molecular switches. However, the underlying thermodynamic design principles that drive their fold-switching behaviour is poorly understood. Gaining insights into the molecular driving forces leading to fold switching behavior is crucial for the rational design of new metamorphic proteins based molecular switches, sensors and stimulus responsive nanomaterials. In this study, we perform a detailed thermodynamic analysis of a designed fold-switching protein [PNAS 2023, 120 (4), e2215418120] 1 that transitions between a 3 fold and /{beta} fold upon changes in temperature. We use an efficient advanced sampling molecular simulation based free energy calculation approach called Confine-Desolvate-Convert-Solvate-Release (CDCSR), which subjects the protein through the complete range of thermodynamic cycle and deconvolutes the enthalpic and entropic driving forces at each stage of the cycle. We find that while 3 fold is stabilized at low temperatures by enthalpic contributions from favorable water-water and protein-water interactions, the transition to the /{beta} fold at high temperatures is driven by the gain of entropy from the release of ordered water molecules surrounding the 3 fold. Our study elucidates the molecular driving forces governing temperature-induced fold-switching behavior and provides a rigorous statistical thermodynamic framework that can help in the design and engineering of future synthetic and functional metamorphic proteins. SignificanceRecent success in the de novo protein design for molecular function is powered by the transformative AI methods and interpreted using the classical wisdom from the Physics-based modelling approaches. Stimuli-sensitive fold switching proteins that can take multiples shapes based on environmental factors are the next frontier in de novo protein engineering. In this work, we unravel the thermodynamic driving forces behind a recently designed temperature-sensitive artificial protein and provide a Physics-based framework to understand the design principles leading to fold switching behavior. Our work compliments the ongoing AI methods that are being explored to unravel the hidden evolutionary embeddings inherent in fold-switching proteins and also highlights the importance of thinking in terms of entropy-based design principles for natural system.

The uracil:uracil (U:U) base pair is one of the most common mismatches observed in RNA. It is notable for its ability to adopt multiple conformational states depending on its structural environment, particularly on the identity of the flanking canonical base pairs. Here, we employed extensive molecular dynamics (MD) simulations to systematically investigate the conformational dynamics of a U:U mismatch embedded within a model A-form RNA helix, flanked by all possible canonical base pair combinations. We found that the neighboring base pairs strongly influence the preferred conformational states of the U:U mismatch. However, the mismatch still regularly samples the less favored conformations on a timescale of hundreds of nanoseconds. Contrary to previous assumptions, water-mediated conformations are not universally the most stable conformational states for isolated U:U mismatches as some of the variants distinctly prefer the direct H-bonding while others destabilize the U:U mismatch altogether. Our results strongly suggest that the presence of a U:U mismatch introduces local strain into the RNA helix, which can be relieved through dynamic destabilization of either the mismatch itself or the flanking canonical base pairs, occasionally forming a shifting "bubble" of instability. These findings advance our understanding of U:U mismatch behavior in RNA and reveal a complex interplay between local sequence context, structural stability, and RNA dynamics.

Fresh two-dimensional (2D) molybdenum disulfide (MoS2) can absorb the hydrocarbon contamination from the ambient air and cause surface aging. Thus, understanding how the surface aging process of MoS2 affects the interaction with biomolecules is crucial for its applications in the biomedical field. Here, we employed atomistic molecular dynamics simulations to investigate the interactions of fresh and aged MoS2 nanosheets with POPE lipid membranes. Our results show that even though both the fresh and aged MoS2 nanosheets are capable of spontaneous insertion into the POPE bilayer membrane, the fresh MoS2 nanosheet displays significantly more robust interaction than the aged one. The potential mean force (PMF) calculations further confirm that the fresh MoS2 nanosheet is more energetically favorable than the aged one in penetrating into the POPE lipid membranes, with the former having ~17 kJ/mol stronger binding affinity than the later. This work provides a deeper understanding of the surface-aging-dependent interaction of MoS2 nanosheet with biomolecules, which might help the design of better MoS2-based nanodevices with appropriate surface properties.

Establishing the fundamental relationships between peptide sequences and fibril formation is critical both for understanding protein misfolding processes and for guiding biomaterial design. Here, we combine all-atom molecular dynamics (MD) simulations with artificial intelligence (AI) to investigate how subtle variations in the arrangement of a short peptide sequence affect its propensity to form fibrils. Our results show that small shifts in the distribution of hydrophobic residues and charge clusters can significantly influence both the nucleation rate and the stability of cross-{beta} structures. To rapidly extend this analysis over a wide sequence space, we developed an active learning-enhanced framework--Machine Learning for Molecular Dynamics (ML4MD)--that iteratively refines its predictions based on MD-derived aggregation data. ML4MD efficiently screens numerous peptide permutations and guides the discovery of previously unrecognized fibril-prone sequences, achieving an area under the receiver operating characteristic (ROC) curve (AUC) of 0.939. Overall, ML4MD streamlines the rational design of amyloid-like peptides by integrating detailed atomistic simulations with rapid and high-accuracy ML predictions.

Riboswitches are non-coding RNA that regulate gene expression by folding into specific three-dimensional structures (holo-form) upon binding by their cognate ligand in the presence of Mg2+. Riboswitch functioning is also hypothesized to be under kinetic control requiring large cognate ligand concentrations. We ask the question under thermodynamic conditions, can the riboswitches populate holo-form like structures in the absence of their cognate ligands only in the presence of Mg2+. We addressed this question using thiamine pyrophosphate (TPP) riboswitch as a model system and computer simulations using a coarse-grained model for RNA. The folding free energy surface (FES) shows that with the initial increase in Mg2+ concentration ([Mg2+]), TPP AD undergoes a barrierless collapse in its dimensions. On further increase in [Mg2+], intermediates separated by barriers appear on the FES, and one of the intermediates has a TPP ligand-binding competent structure. We show that site-specific binding of the Mg2+ aids in the formation of tertiary contacts. For [Mg2+] greater than physiological concentration, AD folds into its holo-form like structure even in the absence of the TPP ligand. The folding kinetics shows that it populates an intermediate due to the misalignment of the two arms in the TPP AD, which acts as a kinetic trap leading to larger folding timescales. The predictions of the intermediate structures from the simulations are amenable for experimental verification.

Magnesium ions (Mg{superscript 2}) play a critical role in RNA structure stabilization by forming various coordinated complexes, preferentially interacting with the backbone phosphate groups. Using extensive atomistic and free energy simulations across simple models and RNA structures of varying complexity, we characterized critical components of the RNA-ion-atmosphere. Radial distribution function analysis reveals distinct peak positions for direct (inner) and solvent-separated (outer-sphere) Mg2+-phosphate coordination layers, aligning with solution X-ray diffraction data. Addressing forcefield limitations, the free energy calculations quantify the kinetic barriers for Mg{superscript 2}-phosphate binding, benchmarking parameters against {superscript 2}Mg NMR measurement. Free energy calculations further explore Mg{superscript 2} chelation with bi-phosphate coordinated Mg2+ systems, identifying a dynamic ensemble of pre-chelate complexes, in addition to a chelated and outer-sphere hexa-hydrated state of Mg2+. In the pre-chelated states, Mg{superscript 2} maintains one inner-sphere interaction while simultaneously coordinating with multiple other phosphates in a solvent-separated manner, referred to as meta-sphere coordination. The pre-chelated complexes from different solvents-separated layers undergo a frequent transition and mediate a unique oxygen exchange mechanism between phosphate and water ligands. Insights into the free energy landscape of SAM-I RNA aptamer further emphasize the significance of pre-chelate complexes for complex RNA structure stabilization, where a number of such solvent-separated dynamic phosphate groups are found to influence Mg2+-RNA coordination. The comprehensive thermodynamic analysis of Mg{superscript 2} chelation and quantitative characterizations of various RNA-ion coordination modes, including this new meta-sphere coordination, provides vital insights for advancing RNA modelling and experimental exploration of complex phosphate networks in the RNA structures.

Short, cysteine-rich peptides can exist in stable or metastable structural ensembles due to the number of possible patterns of formation of their disulfide bonds. One interesting subset of this peptide group is the coonotoxins, which are produced by aquatic snails in the family Conidae. The {micro} conotoxins, which are antagonists and blockers of the voltage-gated sodium channel, exist in a folding spectrum: on one end of the spectrum are more hirudin-like folders, which form disulfide bonds and then reshuffle them, leading to an ensemble of kinetically trapped isomers-and on the other end are more BPTI-like folders-which form the native disulfide bonds one by one in a particular order, leading to a preponderance of conformations existing in a single stable state. In this article, we employ the composite diffusion map approach to study the unified free energy surface of pre-folding {micro}-conotoxin equilibrium. We identify the two most important nonlinear collective modes of the unified folding landscape and demonstrate that in the absence of their disulfides, the conotoxins can be thought of as largely disordered polymers. A small increase in the number of hydrophobic residues in the protein shifts the free energy landscape towards hydrophobically collapsed coil conformations responsible for cysteine proximity in hirudin-like folders, compared to semi-extended coil conformations with more distal cysteines in BPTI-like folders. Overall, this work sheds important light on the folding processes and free energy landscapes of cysteinerich peptides and demonstrates the extent to which sequence and length contribute to these landscapes.

The mechanism of protein stabilization by zwiterionic osmolytes has remained a long-standing puzzle. While the prevalent mechanistic hypothesis suggests an osmo-phobic model in which osmolytes are assumed to stabilize proteins by preferentially excluding themselves from the protein surface, emerging evidences of preferential binding of popular osmolyte trimethyl amine N-oxide (TMAO) with hydrophobic macromolecules contradict this view. Here we address these contrasting perspectives by investigating the folding mechanism of a set of mini proteins in aqueous solutions of two different osmolytes glycine and TMAO, via free energy simulations. Our results demonstrate that, while both osmolytes are found to stabilize the folded conformation of the mini proteins, their mechanism of actions are mutually diverse: Specifically, glycine always depletes from the surface of all mini proteins, thereby conforming to the osmophobic model; but TMAO is found to display ambivalent signatures of protein-specific preferential binding and exclusion to/from the protein surface. At molecular level, the presence of an extended hydrophobic patch in protein topology is found to be recurrent motif in proteins leading to favorable binding with TMAO. Finally, an analysis based upon the preferential interaction theory and folding free energetics reveals that irrespective of preferential binding vs exclusion of osmolytes, it is the relative preferential depletion of osmolytes on transition from folded to unfolded conformation of proteins, which drives the overall conformational equilibrium towards the folded state in presence of osmolytes. Taken together, moving beyond the model system and hypothesis, this work brings out ambivalent mechanism of osmolytes on proteins and provides an unifying justification.

The microscopic rate constants that govern an enzymatic reaction are only directly measured under certain experimental set-ups, such as stopped flow, quenched flow, or temperaturejump assays; the majority of enzymology proceeds from steady state conditions which leads to a set of more easily-observable parameters such as kcat, KM, and observed Kinetic Isotope Effects [Formula]. This paper further develops a model from Toney (2013) to estimate microscopic rate constants from steady-state data for a set of reversible, four-step reactions. This paper uses the Bayesian modeling software Stan, and demonstrates the benefits of Bayesian data analysis in the estimation of these rate constants. In contrast to the optimization methods employed often in the estimation of kinetic constants, a Bayesian treatment is more equipped to estimate the uncertainties of each parameter; sampling from the posterior distribution using Hamiltonian Monte Carlo immediately gives parameter estimates as mean or median of the posterior, and also confidence intervals that express the uncertainty of each parameter.

The outer membrane of bacteria is known to play an important role in the rapid response to desiccation, although the causes and the extent of these effects are still mostly unclear. For this reason, in this work we study the desiccation response of the Gram-negative lipopolysaccha-ride (LPS) bacterial outer membranes. By analyzing molecular dynamics simulations of LPS membranes of different composition during desiccation, we identified the formation of a rigid protective layer of polysaccharides that not only reduces the water evaporation but is also able to indirectly preserve several structural features and as such membrane functionality. Overall, we found that the presence of polysaccharide layer is critical in the conservation of a layer of water in proximity of the hydrophobic region as well as the structure of the lipids acyl chain structure.