Transcription-induced active forces suppress chromatin motion.

The organization of interphase chromosomes in a number of species is starting to emerge thanks to advances in a variety of experimental techniques. However, much less is known about the dynamics, especially in the functional states of chromatin. Some experiments have shown that the motility of individual loci in human interphase chromosome decreases during transcription and increases upon inhibiting transcription. This is a counterintuitive finding because it is thought that the active mechanical force (F) on the order of ten piconewtons, generated by RNA polymerase II (RNAPII) that is presumably transmitted to the gene-rich region of the chromatin, would render it more open, thus enhancing the mobility. We developed a minimal active copolymer model for interphase chromosomes to investigate how F affects the dynamical properties of chromatin. The movements of the loci in the gene-rich region are suppressed in an intermediate range of F and are enhanced at small F values, which has also been observed in experiments. In the intermediate F, the bond length between consecutive loci increases, becoming commensurate with the distance at the minimum of the attractive interaction between nonbonded loci. This results in a transient disorder-to-order transition, leading to a decreased mobility during transcription. Strikingly, the F-dependent change in the locus dynamics preserves the organization of the chromosome at [Formula: see text]. Transient ordering of the loci, which is not found in the polymers with random epigenetic profiles, in the gene-rich region might be a plausible mechanism for nucleating a dynamic network involving transcription factors, RNAPII, and chromatin.

Odd-even disparity in the population of slipped hairpins in RNA repeat sequences with implications for phase separation.

Low-complexity nucleotide repeat sequences, which are implicated in several neurological disorders, undergo liquid-liquid phase separation (LLPS) provided the number of repeat units, n, exceeds a critical value. Here, we establish a link between the folding landscapes of the monomers of trinucleotide repeats and their propensity to self-associate. Simulations using a coarse-grained Self-Organized Polymer (SOP) model for (CAG)n repeats in monovalent salt solutions reproduce experimentally measured melting temperatures, which are available only for small n. By extending the simulations to large n, we show that the free-energy gap, ΔGS, between the ground state (GS) and slipped hairpin (SH) states is a predictor of aggregation propensity. The GS for even n is a perfect hairpin (PH), whereas it is a SH when n is odd. The value of ΔGS (zero for odd n) is larger for even n than for odd n. As a result, the rate of dimer formation is slower in (CAG)30 relative to (CAG)31, thus linking ΔGS to RNA-RNA association. The yield of the dimer decreases dramatically, compared to the wild type, in mutant sequences in which the population of the SH decreases substantially. Association between RNA chains is preceded by a transition to the SH even if the GS is a PH. The finding that the excitation spectrum-which depends on the exact sequence, n, and ionic conditions-is a predictor of self-association should also hold for other RNAs (mRNA for example) that undergo LLPS.

Watching ion-driven kinetics of ribozyme folding and misfolding caused by energetic and topological frustration one molecule at a time.

Folding of ribozymes into well-defined tertiary structures usually requires divalent cations. How Mg2+ ions direct the folding kinetics has been a long-standing unsolved problem because experiments cannot detect the positions and dynamics of ions. To address this problem, we used molecular simulations to dissect the folding kinetics of the Azoarcus ribozyme by monitoring the path each molecule takes to reach the folded state. We quantitatively establish that Mg2+ binding to specific sites, coupled with counter-ion release of monovalent cations, stimulate the formation of secondary and tertiary structures, leading to diverse pathways that include direct rapid folding and trapping in misfolded structures. In some molecules, key tertiary structural elements form when Mg2+ ions bind to specific RNA sites at the earliest stages of the folding, leading to specific collapse and rapid folding. In others, the formation of non-native base pairs, whose rearrangement is needed to reach the folded state, is the rate-limiting step. Escape from energetic traps, driven by thermal fluctuations, occurs readily. In contrast, the transition to the native state from long-lived topologically trapped native-like metastable states is extremely slow. Specific collapse and formation of energetically or topologically frustrated states occur early in the assembly process.

Moderate activity of RNA chaperone maximizes the yield of self-spliced pre-RNA in vivo.

CYT-19 is a DEAD-box protein whose adenosine-triphosphate (ATP)-dependent helicase activity facilitates the folding of group I introns in precursor RNA (pre-RNA) of Neurospora crassa (N. crassa). In the process, they consume a substantial amount of ATP. While much of the mechanistic insight into CYT-19 activity has been gained through the studies on the folding of Tetrahymena group I intron ribozyme, the more biologically relevant issue, namely the effect of CYT-19 on the self-splicing of pre-RNA, remains largely unexplored. Here, we employ a kinetic network model, based on the generalized iterative annealing mechanism (IAM), to investigate the relation between CYT-19 activity, rate of ribozyme folding, and the kinetics of the self-splicing reaction. The network rate parameters are extracted by analyzing the recent biochemical data for CYT-19-facilitated folding of Tetrahymena ribozyme. We then build extended models to explore the metabolism of pre-RNA. We show that the timescales of chaperone-mediated folding of group I ribozyme and self-splicing reaction compete with each other. As a consequence, in order to maximize the self-splicing yield of group I introns in pre-RNA, the chaperone activity must be sufficiently large to unfold the misfolded structures, but not too large to unfold the native structures prior to the self-splicing event. We discover that despite the promiscuous action on structured RNAs, the helicase activity of CYT-19 on group I ribozyme gives rise to self-splicing yields that are close to the maximum.

Information flow, gating, and energetics in dimeric molecular motors.

Molecular motors, kinesin and myosin, are dimeric consisting of two linked identical monomeric globular proteins. Fueled by the free energy generated by ATP hydrolysis, they walk on polar tracks (microtubule or filamentous actin) processively, which means that only one head detaches and executes a mechanical step while the other stays bound to the track. One motor head must regulate the chemical state of the other, referred to as "gating", a concept that is still not fully understood. Inspired by experiments, showing that only a fraction of the energy from ATP hydrolysis is used to advance the kinesin motors against load, we demonstrate that the rest of the energy is associated with chemical transitions in the two heads. The coordinated chemical transitions involve communication between the two heads - a feature that characterizes gating. We develop a general framework, based on information theory and stochastic thermodynamics, and establish that gating could be quantified in terms of information flow between the motor heads. Applications to kinesin-1 and Myosin V show that information flow, with positive cooperativity, at external resistive loads less than a critical value, Fc. When force exceeds Fc, effective information flow ceases. Interestingly, Fc, which is independent of the input energy generated through ATP hydrolysis, coincides with the force at which the probability of backward steps starts to increase. Our findings suggest that transport efficiency is optimal only at forces less than Fc, which implies that these motors must operate at low loads under in vivo conditions.

Formation of extramembrane ß-strands controls dimerization of transmembrane helices in amyloid precursor protein C99.

The 99-residue C-terminal domain of amyloid precursor protein (APP-C99), precursor to amyloid beta (Aß), is a transmembrane (TM) protein containing intrinsically disordered N- and C-terminal extramembrane domains. Using molecular dynamics (MD) simulations, we show that the structural ensemble of the C99 monomer is best described in terms of thousands of states. The C99 monomer has a propensity to form ß-strand in the C-terminal extramembrane domain, which explains the slow spin relaxation times observed in paramagnetic probe NMR experiments. Surprisingly, homodimerization of C99 not only narrows the conformational ensemble from thousands to a few states through the formation of metastable ß-strands in extramembrane domains but also stabilizes extramembrane α-helices. The extramembrane domain structure is observed to dramatically impact the homodimerization motif, resulting in the modification of TM domain conformations. Our study provides an atomic-level structural basis for communication between the extramembrane domains of the C99 protein and TM homodimer formation. This finding could serve as a general model for understanding the influence of disordered extramembrane domains on TM protein structure.

Conformational fluctuations and phases in fused in sarcoma (FUS) low-complexity domain.

The well-known phenomenon of phase separation in synthetic polymers and proteins has become a major topic in biophysics because it has been invoked as a mechanism of compartment formation in cells, without the need for membranes. Most of the coacervates (or condensates) are composed of Intrinsically Disordered Proteins (IDPs) or regions that are structureless, often in interaction with RNA and DNA. One of the more intriguing IDPs is the 526-residue RNA-binding protein, Fused in Sarcoma (FUS), whose monomer conformations and condensates exhibit unusual behavior that are sensitive to solution conditions. By focussing principally on the N-terminus low-complexity domain (FUS-LC comprising residues 1-214) and other truncations, we rationalize the findings of solid-state NMR experiments, which show that FUS-LC adopts a non-polymorphic fibril structure (core-1) involving residues 39-95, flanked by fuzzy coats on both the N- and C-terminal ends. An alternate structure (core-2), whose free energy is comparable to core-1, emerges only in the truncated construct (residues 110-214). Both core-1 and core-2 fibrils are stabilized by a Tyrosine ladder as well as hydrophilic interactions. The morphologies (gels, fibrils, and glass-like) adopted by FUS seem to vary greatly, depending on the experimental conditions. The effect of phosphorylation is site-specific. Simulations show that phosphorylation of residues within the fibril has a greater destabilization effect than residues that are outside the fibril region, which accords well with experiments. Many of the peculiarities associated with FUS may also be shared by other IDPs, such as TDP43 and hnRNPA2. We outline a number of problems for which there is no clear molecular explanation.

A Review on Small Organic Colorimetric and Fluorescent Hosts for the Detection of Cobalt and Nickel Ion.

In recent years, there has been a notable increase in efforts to advance efficient hosts for detecting cobalt and nickel ions, driven by their extensive industrial applications and environmental significance. This review meticulously examines the progress made in small organic colorimetric and fluorescent hosts tailored specifically for the sensitive and selective detection of cobalt and nickel ions. It delves into a diverse range of molecular architectures, including organic ligands, elucidating their unique attributes such as sensitivity, selectivity, and response time. Moreover, the review precisely explores the underlying principles governing the colorimetric and fluorescent mechanisms employed by these hosts, shedding light on the intricate interactions between the sensing moieties and the target metal ions. Furthermore, it critically evaluates the practical applicability of these hosts, considering crucial factors such as detection limits, recyclability, and compatibility with complex sample matrices. Additionally, exploration extends to potential challenges and prospects in the field, emphasizing the imperative for ongoing innovation to address emerging environmental and analytical demands. Eventually, through this comprehensive examination, the review seeks to contribute to the ongoing endeavor to develop robust and efficient tools for monitoring and detecting cobalt and nickel metal ions in diverse analytical scenarios.

Scaling regimes for wormlike chains confined to cylindrical surfaces under tension.

We compute the free energy of confinement [Formula: see text] for a wormlike chain (WLC), with persistence length [Formula: see text], that is confined to the surface of a cylinder of radius R under an external tension f using a mean field variational approach. For long chains, we analytically determine the behavior of the chain in a variety of regimes, which are demarcated by the interplay of [Formula: see text], the Odijk deflection length ([Formula: see text]), and the Pincus length ([Formula: see text], with [Formula: see text] being the thermal energy). The theory accurately reproduces the Odijk scaling for strongly confined chains at [Formula: see text], with [Formula: see text]. For moderate values of f, the Odijk scaling is discernible only when [Formula: see text] for strongly confined chains. Confinement does not significantly alter the scaling of the mean extension for sufficiently high tension. The theory is used to estimate unwrapping forces for DNA from nucleosomes.

Shape changes and cooperativity in the folding of the central domain of the 16S ribosomal RNA.

Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg2+ ions or by interaction with ribosomal proteins. To provide a quantitative description of ion-induced folding of the ∼350-nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg2+ concentration between 0 and 30 mM. The Mg2+ dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg2+ concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg2+ ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg2+-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg2+-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome.

Universal Nature of Collapsibility in the Context of Protein Folding and Evolution.

Theory and simulations predicted that the sizes of the unfolded states of globular proteins should decrease as the denaturant concentration is reduced from a high to a low value. However, small angle X-ray scattering (SAXS) data were used to assert the opposite, while interpretation of single molecule Förster resonance energy transfer experiments (FRET) supported the theoretical predictions. The disagreement between the two experiments is the SAXS-FRET controversy. By harnessing recent advances in SAXS and FRET experiments and setting these findings in the context of a general theory and simulations, which do not rely on experimental data, we establish that compaction of unfolded states under native conditions is universal. The theory also predicts that proteins rich in ß-sheets are more collapsible than α-helical proteins. Because the extent of compaction is small, experiments have to be accurate and their interpretations should be as model-free as possible. Theory also suggests that collapsibility itself could be a physical restriction on the evolution of foldable sequences, and also provides a physical basis for the origin of multidomain proteins.

Entropic contribution of ACE2 glycans to RBD binding.

The spike protein of the SARS-CoV-2 virus (the causative agent of COVID-19) recognizes the host cell by binding to the peptidase domain (PD) of the extracellular receptor angiotensin-converting enzyme 2 (ACE2). A variety of carbohydrates could be attached to the six asparagines in the PD, resulting in a heterogeneous population of ACE2 glycoforms. Experiments have shown that the binding affinity of glycosylated and deglycosylated ACE2 to the virus is virtually identical. In most cases, the reduction in glycan size correlates with stronger binding, which suggests that volume exclusion, and hence entropic forces, determine the binding affinity. Here, we quantitatively test the entropy-based hypothesis by developing a lattice model for the complex between ACE2 and the SARS-CoV-2 spike protein receptor-binding domain (RBD). Glycans are treated as branched polymers with only volume exclusion, which we justify using all-atom molecular dynamics simulations in explicit water. We show that the experimentally measured changes in the ACE2-RBD dissociation constants for a variety of engineered ACE2 glycoforms are in reasonable agreement with our theory, thus supporting our hypothesis. However, a quantitative recovery of all the experimental data could require weak attractive interactions.

Role of water-bridged interactions in metal ion coupled protein allostery.

Allosteric communication between distant parts of proteins controls many cellular functions, in which metal ions are widely utilized as effectors to trigger the allosteric cascade. Due to the involvement of strong coordination interactions, the energy landscape dictating the metal ion binding is intrinsically rugged. How metal ions achieve fast binding by overcoming the landscape ruggedness and thereby efficiently mediate protein allostery is elusive. By performing molecular dynamics simulations for the Ca2+ binding mediated allostery of the calmodulin (CaM) domains, each containing two Ca2+ binding helix-loop-helix motifs (EF-hands), we revealed the key role of water-bridged interactions in Ca2+ binding and protein allostery. The bridging water molecules between Ca2+ and binding residue reduces the ruggedness of ligand exchange landscape by acting as a lubricant, facilitating the Ca2+ coupled protein allostery. Calcium-induced rotation of the helices in the EF-hands, with the hydrophobic core serving as the pivot, leads to exposure of hydrophobic sites for target binding. Intriguingly, despite being structurally similar, the response of the two symmetrically arranged EF-hands upon Ca2+ binding is asymmetric. Breakage of symmetry is needed for efficient allosteric communication between the EF-hands. The key roles that water molecules play in driving allosteric transitions are likely to be general in other metal ion mediated protein allostery.

Observe without disturbing: tracer particles sense local stresses in cell collectives without affecting the cancer cell dynamics.

Measurements of local stresses on the cancer cells (CCs), inferred by embedding inert compressible tracer particles (TPs) in a growing multicellular spheroid (MCS), show that pressure decreases monotonically as the distance from the core of the MCS increases. How faithfully do the TPs report the local stresses in the CCs is an important question because pressure buildup in the MCS is dynamically generated due to CC division, which implies that the CC dynamics should be minimally altered by the TPs. Here using theory and simulations, we show that although the TP dynamics is unusual, exhibiting sub-diffusive behavior on times less than the CC division times and hyper-diffusive dynamics in the long-time limit, they do not affect the long-time CC dynamics. The CC pressure profile within the MCS, which decays from a high value at the core to the periphery, is almost identical with and without the TPs. That the TPs have a small effect on the local stresses in the MCS implies that they are reasonale reporters of the CC microenvironment.

Allosteric communication between ACE2 active site and binding interface with SARS-CoV-2.

SARS-CoV-2, the virus causing COVID-19, initiates cell invasion by deploying a receptor binding domain (RBD) to recognize the host transmembrane peptidase angiotensin-converting enzyme 2 (ACE2). Numerous experimental and theoretical studies have adopted high-throughput and structure-guided approaches to (i) understand how the RBD recognizes ACE2, (ii) rationalize, and (iii) predict the effect of viral mutations on the binding affinity. Here, we investigate the allosteric signal triggered by the dissociation of the ACE2-RBD complex. To this end, we construct an Elastic Network Model (ENM), and we use the Structural Perturbation Method (SPM). Our key result is that complex dissociation opens the ACE2 substrate-binding cleft located away from the interface and that fluctuations of the ACE2 binding cleft are facilitated by RBD binding. These and other observations provide a structural and dynamical basis for the influence of SARS-CoV-2 on ACE2 enzymatic activity. In addition, we identify a conserved glycine (G502 in SARS-CoV-2) as a key participant in complex disassembly.

Asymmetry in histone rotation in forced unwrapping and force quench rewrapping in a nucleosome.

Single molecule pulling experiments have shown that DNA in the nucleosomes unwraps in two stages from the histone protein core (HPC). The first stage, attributed to the rupture of the outer DNA turn, occurs between 3 and 5 pNs, and is reversible. The inner DNA turn ruptures irreversibly at forces between 9 and 15 pNs (or higher) in the second stage. Molecular simulations using the Self-Organized Polymer model capture the experimental findings. The unwrapping of the outer DNA turn is independent of the pulling direction. The rupture of the DNA inner turn depends on the pulling direction and involves overcoming substantial energetic (most likely electrostatic in origin) and kinetic barriers. They arise because the mechanical force has to generate sufficient torque to rotate the HPC by 180°. On the other hand, during the rewrapping process, HPC rotation is stochastic, with force playing no role. The assembly of the outer DNA wrap upon force quench nearly coincides with the unwrapping process, confirming the reversibility of the outer turn rupture. The asymmetry in HPC rotation during unwrapping and rewrapping explains the observed hysteresis in the stretch-release cycles in experiments. We propose experiments to test the prediction that HPC rotation produces kinetic barriers in the unwrapping process.

Differences in the free energies between the excited states of Aß40 and Aß42 monomers encode their aggregation propensities.

The early events in the aggregation of the intrinsically disordered peptide, amyloid-ß (Aß), involve transitions from the disordered free energy ground state to assembly-competent states. Are the fingerprints of order found in the amyloid fibrils encoded in the conformations that the monomers access at equilibrium? If so, could the enhanced aggregation rate of Aß42 compared to Aß40 be rationalized from the sparsely populated high free energy states of the monomers? Here, we answer these questions in the affirmative using coarse-grained simulations of the self-organized polymer-intrinsically disordered protein (SOP-IDP) model of Aß40 and Aß42. Although both the peptides have practically identical ensemble-averaged properties, characteristic of random coils (RCs), the conformational ensembles of the two monomers exhibit sequence-specific heterogeneity. Hierarchical clustering of conformations reveals that both the peptides populate high free energy aggregation-prone ([Formula: see text]) states, which resemble the monomers in the fibril structure. The free energy gap between the ground (RC) and the [Formula: see text] states of Aß42 peptide is smaller than that for Aß40. By relating the populations of excited states of the two peptides to the fibril formation time scales using an empirical formula, we explain nearly quantitatively the faster aggregation rate of Aß42 relative to Aß40. The [Formula: see text] concept accounts for fibril polymorphs, leading to the prediction that the less stable [Formula: see text] state of Aß42, encoding for the U-bend fibril, should form earlier than the structure with the S-bend topology, which is in accord with Ostwald's rule rationalizing crystal polymorph formation.

Chromatin dynamics controls epigenetic domain formation.

In multicellular organisms, nucleosomes carry epigenetic information that defines distinct patterns of gene expression, which are inherited over multiple generations. The enhanced capacity for information storage arises by nucleosome modifications, which are triggered by enzymes. Modified nucleosomes can transfer the mark to others that are in proximity by a positive-feedback (modification begets modification) mechanism. We created a generic polymer model, referred to as 3DSpreader, in which each bead, representing a nucleosome, stochastically switches between unmodified (U) and modified (M) states depending on the states of the neighbors. Modification begins at a specific nucleation site (NS) that is permanently in the M state, and could spread to other loci that is dictated by chromatin dynamics. Transfer of marks among the non-nucleation loci occurs stochastically as chromatin evolves in time. If the spreading rate is slower than the chromatin relaxation rate, which is biologically pertinent, then finite-sized domains form, driven by contacts between nucleosomes through a three-dimensional looping mechanism. Surprisingly, simulations based on the 3DSpreader model result in finite bounded domains that arise without the need for any boundary elements. Maintenance of spatially and temporally stable domains requires the presence of the NS, whose removal eliminates finite-sized modified domains. The theoretical predictions are in excellent agreement with experimental data for H3K9me3 spreading in mouse embryonic stem cells.

Adhesion strength between cells regulate nonmonotonic growth by a biomechanical feedback mechanism.

We determine how intercellular interactions and mechanical pressure experienced by single cells regulate cell proliferation using a minimal computational model for three-dimensional multicellular spheroid (MCS) growth. We discover that emergent spatial variations in the cell division rate, depending on the location of the cells either at the core or periphery within the MCS, is regulated by intercellular adhesion strength (fad). Varying fad results in nonmonotonic proliferation of cells in the MCS. A biomechanical feedback mechanism coupling the fad and microenvironment-dependent pressure fluctuations relative to a threshold value (pc) determines the onset of a dormant phase, and explains the nonmonotonic proliferation response. Increasing fad from low values enhances cell proliferation because pressure on individual cells is smaller compared with pc. However, at high fad, cells readily become dormant and cannot rearrange effectively in spacetime, leading to arrested cell proliferation. Utilizing our theoretical predictions, we explain experimental data on the impact of adhesion strength on cell proliferation and find good agreement. Our work, which shows that proliferation is regulated by pressure-adhesion feedback mechanism, may be a general feature of multicellular growth.

Mechanical feedback controls the emergence of dynamical memory in growing tissue monolayers.

The growth of a tissue, which depends on cell-cell interactions and biologically relevant processes such as cell division and apoptosis, is regulated by a mechanical feedback mechanism. We account for these effects in a minimal two-dimensional model in order to investigate the consequences of mechanical feedback, which is controlled by a critical pressure, pc. A cell can only grow and divide if its pressure, due to interaction with its neighbors, is less than pc. Because temperature is not a relevant variable, the cell dynamics is driven by self-generated active forces (SGAFs) that arise due to cell division. We show that even in the absence of intercellular interactions, cells undergo diffusive behavior. The SGAF-driven diffusion is indistinguishable from the well-known dynamics of a free Brownian particle at a fixed finite temperature. When intercellular interactions are taken into account, we find persistent temporal correlations in the force-force autocorrelation function (FAF) that extends over a timescale of several cell division times. The time-dependence of the FAF reveals memory effects, which increases as pc increases. The observed non-Markovian effects emerge due to the interplay of cell division and mechanical feedback and are inherently a non-equilibrium phenomenon.