Melting and Bubble Formation in a Double-Stranded DNA: Microscopic Aspects of Early Base-Pair Opening Events and the Role of Water.

Despite its rigid structure, DNA is a remarkably flexible molecule. Flexibility is essential for biological functions (such as transcription and gene repair), which require large-amplitude structural changes such as bubble formation. The bubbles thus formed are required to have a certain stability of their own and survive long on the time scale of molecular motions. A molecular understanding of fluctuations leading to quasi-stable structures is not available. Through extensive atomistic molecular dynamics simulations, we identify a sequence of microscopic events that culminate in local bubble formation, which is initiated by base-pair (BP) opening, resulting from the cleavage of native BP hydrogen bonds (HBs). This is followed by the formation of mismatched BPs with non-native contacts. These metastable structures can either revert to their original forms or undergo a flipping transition to form a local bubble that can span across 3-4 BPs. A substantial distortion of the DNA backbone and a disruption of BP stacking are observed because of the structural changes induced by these local perturbations. We also explored how water helps in the entire process. A small number of water molecules undergo rearrangement to stabilize the intermediate states by forming HBs with DNA bases. Water thus acts as a lubricant that counteracts the enthalpic penalty suffered from the loss of native BP contacts. Although the process of bubble formation is reversible, the sequence of steps involved poses an entropic barrier, preventing it from easily retracing the path to the native state.

Sequence Sensitivity in Membrane Remodeling by Polyampholyte Condensates.

Intrinsically disordered peptides (IDPs) have been found to undergo liquid-liquid phase separation (LLPS) and produce complex coacervates that play numerous regulatory roles in the cell. Recent experimental studies have discovered that LLPS at or near the membrane surface helps in the biomolecular organization during signaling events and can significantly alter the membrane morphology. However, the molecular mechanism and microscopic details of such processes still remain unclear. Here we study the effect of polyampholyte and polyelectrolyte condensation on two different anionic membranes, as they represent a majority of naturally occurring IDPs. The polyampholytes are fifty-residue polymers, made of glutamate(E) and lysine(K) with different charge patterns. The polyelectrolytes are separate chains of E25 and K25. We first calibrate the MARTINI v3.0 force field and then perform long-time-scale coarse-grained molecular dynamics simulations. We find that condensates formed by all the polyampholytes get adsorbed on the membrane. However, the strong polyampholytes (i.e., blocky sequences) can remodel the membranes more prominently than the weaker ones (i.e., scrambled sequences). Condensates formed by the blocky sequences induce a significant negative curvature (∼0.1 nm-1) and local demixing of lipids, whereas those by the scrambled sequences tend to wet the membrane to a greater extent without generating significant curvature or demixing. We perform several microscopic analyses to characterize the nature of the interaction between membranes and these condensates. Our analyses of interaction energetics reveal that membrane remodeling and/or wetting are favored by enhanced interactions between polyampholytes with lipids and the counterions.

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation.

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation.

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

Coacervation-Induced Remodeling of Nanovesicles.

Intrinsically disordered peptides can form biomolecular condensates through liquid-liquid phase separation. These condensates play diverse roles in cells, including inducing large-scale changes in membrane morphology. Here we employ coarse-grained molecular dynamics simulations to identify the most salient physical principles that govern membrane remodeling by condensates. By systematically varying the interaction strengths among the polymers and lipids in our coarse-grained model, we are able to recapitulate various membrane transformations observed in different experiments. Endocytosis and exocytosis of the condensate are observed when the interpolymeric attraction is stronger than polymer-lipid interaction. We find a critical size of the condensate required to exhibit successful endocytosis. Multilamellarity and local gelation are observed when the polymer-lipid attraction is significantly stronger than the interpolymeric attraction. Our insights provide essential guidance to the design of (bio)polymers for the manipulation of membrane morphology in various applications such as drug delivery and synthetic biology.

Bimodal 1/f Noise and Anticorrelation between DNA-Water and DNA-Ion Energy Fluctuations.

The coupling between the conformational fluctuations of DNA and its surrounding environment, consisting of water and ions in solution, remains poorly understood and relatively less investigated as compared to proteins. Here, with the help of molecular dynamics simulations and statistical mechanical analyses, we explore the dynamical coupling among DNA, water, and counterions through correlations among respective energy fluctuations in both double- (ds-) and single-stranded (ss-) DNA solutions. Fluctuations in the collective DNA-water and DNA-ion interaction energies are found to be strongly anticorrelated across all the systems. The fluctuations of DNA self-energy, however, are weakly coupled to DNA-water and DNA-ion interactions in ds-DNA. An enhancement of the DNA-water coupling is observed in ss-DNA, where the system is less rigid. All the interaction energies exhibit 1/f noise in their energy power spectra with surprisingly prominent bimodality in the DNA-water and DNA-ion fluctuations. The nature of the energy spectra appears to be indifferent to the relative rigidity of the DNA. We discuss the role of the observed correlations in ion-water motions on a DNA duplex in the experimentally observed anomalous slow dielectric relaxation and solvation dynamics and in furthering our understanding of the DNA energy landscape.

Ethanol exchange between two graphene surfaces in nanoconfined aqueous solution: Rate and mechanism.

We observe, by computer simulations, a remarkable long-distance, rare, but repetitive, exchange of ethanol molecules between two parallel graphene surfaces in nanoconfined, aqueous, ethanol solutions. We compute the rate of exchange as a function of the separation (d) between the two surfaces. We discover that the initiating (or, the launching) step in this exchange is the attainment of an instantaneous orientation of the carbon-oxygen bond vector relative to the graphene surface. This observation led us to construct a two-dimensional free energy surface for this exchange, with respect to two order parameters, namely, (i) the perpendicular distance of ethanol molecule from the graphene surfaces, z, and (ii) the orientation of the O-C bond vector, θ, of the tagged ethanol molecule. For d = 3 nm, the rate of exchange is found to be 0.44 ns-1 for the force field used. We also vary the force field and determine the sensitivity of the rate. From the free energy landscape, one could determine the minimum energy pathway. We use both, the transition state theory and Kramers' theory, to calculate the rate. The calculated rate agrees well with the simulated value as mentioned above. We find that the rate of exchange phenomenon is sensitive to the interaction strength of graphene and the hydrophobic group of ethanol. The free energy landscape exchange shows dependence on the distance separation of the two hydrophobic surfaces and reveals interesting features.

From structure and dynamics to biomolecular functions: The ubiquitous role of solvent in biology.

Biological activity requires a solvent that can provide a suitable environment, which satisfies the twin need for stability and the ability to change. Among all the solvents water plays the most important role. We review, analyze, and comment on recent works on the structure and dynamics of water around biomolecules and their role in specific biological functions. While studies in the past have focused on understanding the biomolecule-water interactions through a hydration layer; recently the attention has shifted towards understanding functions at a molecular level. Such a microscopic understanding clearly requires elucidation of detailed dynamical processes where solvent molecules play an important role. Finally, we comment on the advances made in understanding the role of water inside a biological cell.

Coacervation of poly-electrolytes in the presence of lipid bilayers: mutual alteration of structure and morphology.

Many intrinsically disordered peptides have been shown to undergo liquid-liquid phase separation and form complex coacervates, which play various regulatory roles in the cell. Recent experimental studies found that such phase separation processes may also occur at the lipid membrane surface and help organize biomolecules during signaling events; in some cases, phase separation of proteins at the membrane surface was also observed to lead to significant remodeling of the membrane morphology. The molecular mechanisms that govern the interactions between complex coacervates and lipid membranes and the impacts of such interactions on their structure and morphology, however, remain unclear. Here we study the coacervation of poly-glutamate (E30) and poly-lysine (K30) in the presence of lipid bilayers of different compositions. We carry out explicit-solvent coarse-grained molecular dynamics simulations by using the MARTINI (v3.0) force-field. We find that more than 20% anionic lipids are required for the coacervate to form stable contact with the bilayer. Upon wetting, the coacervate induces negative curvature to the bilayer and facilitates local lipid demixing, without any peptide insertion. The magnitude of negative curvature, extent of lipid demixing, and asphericity of the coacervate increase with the concentration of anionic lipids. Overall, we observe a decrease in the number of contacts among the polyelectrolytes as the droplet spreads over the bilayer. Therefore, unlike previous suggestions, interactions among polyelectrolytes do not constitute a driving force for the membrane bending upon wetting by the coacervate. Rather, analysis of interaction energy components suggests that bending of the membrane is favored by enhanced interactions between polyelectrolytes with lipids as well as with counterions. Kinetic studies reveal that, at the studied polyelectrolyte concentrations, the coacervate formation precedes bilayer wetting.

An initiative to reduce medication errors in neonatal care unit of a tertiary care hospital, Kolkata, West Bengal: a quality improvement report.

BACKGROUND: Medication errors are an emerging problem in various hospital settings, especially in neonates. A study conducted in the neonatal care unit of a tertiary institute in Kolkata as baseline over 3 months, revealed total error to be around 71.1/100 prescriptions (median medication error percentage: 63%). PURPOSE: To assess the occurrences of medication errors and determine efficacy of Point-of-Care Quality improvement (POCQI) model in reducing the same from baseline 63% to less than 10%, in the above setting within next 9 months. MATERIALS AND METHODS: This quality improvement initiative of quasi-experimental design comprised randomly selected prescriptions and monitoring sheets of neonates admitted in the neonatal care unit, obeying inclusion and exclusion criteria. Medication errors were assessed and categorised using a predesigned and pretested checklist. Interventions were planned after forming a quality improvement team in four plan-do-study-act (PDSA) cycles spanning over 6 weeks each (including training of doctors and nurses, signature and countersignatures of respective healthcare personnel, computer-generated prescriptions and newly designed software-generated prescriptions) as per POCQI model of the WHO and results in post-intervention phase (3 months) were compared. RESULTS: A total of 552 prescriptions and monitoring sheets of 124 neonates were studied. Median medication error percentages in first, second, third and fourth PDSA cycle were, respectively, 48%, 42%, 30% and 14%. Total error reduced to 10.4/100 prescriptions (p<0.005), with significant reduction in erred dosage, timing, interval, preparation and rate of infusion of drugs in prescriptions of the post-intervention phase. CONCLUSION: Implementation of change ideas via PDSA cycles, as per the POCQI model with technological aid, significantly decreased the percentage of medication errors in neonates, which was also sustained in the post-intervention phase and facilitated error-free prescriptions.

Tug-of-War between Internal and External Frictions and Viscosity Dependence of Rate in Biological Reactions.

The role of water in biological processes is studied in three reactions, namely, the Fe-CO bond rupture in myoglobin, GB1 unfolding, and insulin dimer dissociation. We compute both internal and external components of friction on relevant reaction coordinates. In all of the three cases, the cross-correlation between forces from protein and water is found to be large and negative that serves to reduce the total friction significantly, increase the calculated reaction rate, and weaken solvent viscosity dependence. The computed force spectrum reveals bimodal 1/f noise, suggesting the use of a non-Markovian rate theory.

Stochastic formulation of multiwave pandemic: decomposition of growth into inherent susceptibility and external infectivity distributions.

Many known and unknown factors play significant roles in the persistence of an infectious disease, but two that are often ignored in theoretical modelling are the distributions of (i) inherent susceptibility ( σ inh ) and (ii) external infectivity ( ι ext ), in a population. While the former is determined by the immunity of an individual towards a disease, the latter depends on the exposure of a susceptible person to the infection. We model the spatio-temporal propagation of a pandemic as a chemical reaction kinetics on a network using a modified SAIR (Susceptible-Asymptomatic-Infected-Removed) model to include these two distributions. The resulting integro-differential equations are solved using Kinetic Monte Carlo Cellular Automata (KMC-CA) simulations. Coupling between σ inh and ι ext are combined into a new parameter Ω, defined as Ω = σ inh × Î¹ ext ; infection occurs only if the value of Ω is greater than a Pandemic Infection Parameter (PIP), Ω 0 . Not only does this parameter provide a microscopic viewpoint of the reproduction number R0 advocated by the conventional SIR model, but it also takes into consideration the viral load experienced by a susceptible person. We find that the neglect of this coupling could compromise quantitative predictions and lead to incorrect estimates of the infections required to achieve the herd immunity threshold. The figure represents the network model for spread of infectious diseases considered in this work. It also shows the resultant multiwave infection graph by inclusion of inherent susceptibility and external infectivity distributions and migration of infected individuals.

Structural Stability of Insulin Oligomers and Protein Association-Dissociation Processes: Free Energy Landscape and Universal Role of Water.

Association and dissociation of proteins are important biochemical events. In this Feature Article, we analyze the available studies of these processes for insulin oligomers in aqueous solution. We focus on the solvation of the insulin monomer in water, stability and dissociation of its dimer, and structural integrity of the hexamer. The intricate role of water in solvation of the dimer- and hexamer-forming surfaces, in long-range interactions between the monomers and the stability of the oligomers, is discussed. Ten water molecules inside the central cavity stabilize the structure of the insulin hexamer. We discuss how different order parameters can be used to understand the dissociation of the insulin dimer. The calculation of the rate using a recently computed multidimensional free energy provides considerable insight into the interplay between protein and water dynamics.

Rate of Insulin Dimer Dissociation: Interplay between Memory Effects and Higher Dimensionality.

We calculate the rate of dissociation of an insulin dimer into two monomers in water. The rate of this complex reaction is determined by multiple factors that are elucidated. By employing advanced sampling techniques, we first obtain the reaction free energy surface for the dimer dissociation as a function of two order parameters, namely, the distance between the center-of-mass of two monomers (R) and the number of cross-contacts (Q) among the backbone Cα atoms of two monomers. We then construct an orthogonal 2D reaction energy surface by introducing the reaction coordinate X to denote the minimum energy pathway and a conjugate coordinate Y that spans the orthogonal direction. The free energy landscape is rugged with multiple maxima and minima. We calculate the rate by employing not only the non-Markovian multidimensional rate theory but also several other theoretical approaches. The necessary reaction frequencies and the frictions are calculated from the time correlation function formalism. Our best estimate of the rate is 0.4 µs-1. Our study reveals interesting opposite influences of dimensionality and memory in determining the rate constant of the reaction. We gain interesting insights into the dimer dissociation process by looking directly at the trajectories obtained from molecular dynamics simulation.

Unfolding of Dynamical Events in the Early Stage of Insulin Dimer Dissociation.

The dissociation of an insulin dimer is an important biochemical event that could also serve as a prototype of dissociations in similar biomolecular assemblies. We use a recently developed multidimensional free energy landscape for insulin dimer dissociation to unearth the microscopic and mechanistic aspects of the initial stages of the process that could hold the key to understanding the stability and the rate. The following sequence of events occurs in the initial stages: (i) The backbone hydrogen bonds break partially at the antiparallel ß-sheet junction, (ii) the two α-helices (chain B) move away from each other while several residues (chain A) move closer, and (iii) a flow of adjacent water molecules occurs into the junction region. Interestingly, the intermonomeric center-to-center distance does not increase, but the number of native contacts exhibits a sharp decrease. Subsequent steps involve further disengagement of hydrophobic groups. This process is slow because of an entropic bottleneck created by the existence of the large configuration space available in the native state (NS), which is inhabited by low-frequency conformational fluctuations. We carry out a density-of-states analyses in the dimer NS to unearth distinctive features not present in the monomers. These low-frequency modes are also responsible for a large entropic stabilization of the NS. Hydrophobic disengagement in the early stage leads to the formation of a twisted intermediate state which itself is a metastable minimum (IS-1). The subsequent progress leads to another dimeric complex (IS-2), which is on the dissociative pathway and characterized by a further decrease in the native contacts. The dissociation process provides insights into the workings of a biomolecular assembly.

An exact solution in the theory of fluorescence resonance energy transfer with vibrational relaxation.

The elegant expression of Förster that predicts the well-known 1/R6 distance (R) dependence of the rate of energy transfer, although widely used, was derived using several approximations. Notable among them is the neglect of the vibrational relaxation in the reactant (donor) and product (acceptor) manifolds. Vibrational relaxation can play an important role when the energy transfer rate is faster than the vibrational relaxation rate. Under such conditions, donor to acceptor energy transfer can occur from the excited vibrational states. This phenomenon is not captured by the usual formulation based on the overlap of donor emission and acceptor absorption spectra. Here, we develop a Green's function-based generalized formalism and obtain an exact solution for the excited state population relaxation and the rate of energy transfer in the presence of vibrational relaxation. We find that the application of the well-known Förster's expression might lead to overestimation of R.

Anomalous dielectric response of nanoconfined water.

In order to develop a microscopic level understanding of the anomalous dielectric properties of nanoconfined water (NCW), we study and compare three different systems, namely, (i) NCW between parallel graphene sheets (NCW-GSs), (ii) NCW inside graphene covered nanosphere (NCW-Sph), and (iii) a collection of one- and two-dimensional constrained Ising spins with fixed orientations at the termini. We evaluate the dielectric constant and study the scaling of ε with size by using linear response theory and computer simulations. We find that the perpendicular component remains anomalously low at smaller inter-plate separations (d) over a relatively wide range of d. For NCW-Sph, we could evaluate the dielectric constant exactly and again find a low value and a slow convergence to the bulk. To obtain a measure of surface influence into the bulk, we introduce and calculate correlation lengths to find values of ∼9 nm for NCW-GS and ∼5 nm for NCW-Sph, which are surprisingly large, especially for water. We discover that the dipole moment autocorrelations exhibit an unexpected ultrafast decay. We observe the presence of a ubiquitous frequency of ∼1000 cm-1, associated only with the perpendicular component for NCW-GS. This (caging) frequency seems to play a pivotal role in controlling both static and dynamic dielectric responses in the perpendicular direction. It disappears with an increase in d in a manner that corroborates with the estimated correlation length. A similar observation is obtained for NCW-Sph. Interestingly, one- and two-dimensional Ising model systems that follow Glauber spin-flip dynamics reproduce the general characteristics.

Comment on "investigation of dielectric constants of water in a nano-confined pore" by H. Zhu, F. Yang, Y. Zhu, A. Li, W. He, J. Huang and G. Li, RSC Adv., 2020, 10, 8628.

Zhu et al. recently reported the spatially resolved dielectric profile and value of the average static dielectric constant of water confined inside a silica nanopore. However, the authors neglected the inherent anisotropy and non-local nature of the dielectric response under confinement. Neglect of these important issues produces erroneous results and vastly underestimates the average values. We demonstrate the correct way to incorporate the anisotropy and to obtain the average dielectric constant of cylindrically nanoconfined dipolar fluids. Use of the correct theoretical formalism expectedly shows convergence of the calculated dielectric response to the bulk value with increasing the nanopore size. On the contrary, the equation used by Zhu et al. fails to exhibit the convergence of the same. Instead, decreases as the nanopore size is gradually increased.

Water Layer at Hydrophobic Surface: Electrically Dead but Dynamically Alive?

The origin of the anomalous low value of the static dielectric constant (SDC) of confined water has been addressed and unearthed. While the low value is partly due to the different dielectric boundaries, a significant role is played by the "electrically dead layer" (EDL). As the observed dielectric constant is the harmonic mean of the grid-wise SDCs, the first layer, having the smallest SDC, makes a disproportionately large contribution. This enhanced contribution, in turn, arises from the orientationally ordered surface water molecules. They exhibit reduced fluctuations in collective dipole moment, as the molecules remain partly caged due to water-surface interactions. This phenomenon is found to be universal. We study the structure and dynamics of the water molecules which characterize the EDL. We demonstrate that while the EDL remains alive at a molecular level, with a finite residence time, it displays time scales not substantially different compared to the distant water layers.