Search

Several types of molecular machines move along biopolymers like chromatin. However, the details about the microscopic activity of these machines and how to distinguish their modes of action are not well understood. We propose that the activity of such machines can be classified by studying looped chromatin under shear flow. Our simulations show that a chromatin-like polymer with two types of activities-constant (type-I) or local curvature-dependent tangential forces (type-II)-exhibits very different behavior under shear flow. We show that one can distinguish both activities by measuring the nature of a globule-to-extended coil transition, tank treading, and tumbling dynamics.

Cells switch genes ON or OFF by altering the state of chromatin via histone modifications at specific regulatory locations along the chromatin polymer. These gene regulation processes are carried out by a network of reactions in which the histone marks spread to neighboring regions with the help of enzymes. In the literature, this spreading has been studied as a purely kinetic, non-diffusive process considering the interactions between neighboring nucleosomes. In this work, we go beyond this framework and study the spreading of modifications using a reaction-diffusion (RD) model accounting for the diffusion of the constituents. We quantitatively segregate the modification profiles generated from kinetic and RD models. The diffusion and degradation of enzymes set a natural length scale for limiting the domain size of modification spreading, and the resulting enzyme limitation is inherent in our model. We also demonstrate the emergence of confined modification domains without the explicit requirement of a nucleation site. We explore polymer compaction effects on spreading and show that single-cell domains may differ from averaged profiles. We find that the modification profiles from our model are comparable with existing H3K9me3 data of S. pombe., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Manivannan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Knowledge about the dynamic nature of chromatin organization is essential to understand the regulation of processes like DNA transcription and repair. The existing models of chromatin assume that protein organization and chemical states along chromatin are static and the 3D organization is purely a result of protein-mediated intra-chromatin interactions. Here we present a new hypothesis that certain nonequilibrium processes, such as switching of chemical and physical states due to nucleosome assembly/disassembly or gene repression/activation, can also simultaneously influence chromatin configurations. To understand the implications of this inherent nonequilibrium switching, we present a block copolymer model of chromatin, with switching of its segmental states between two states, mimicking active/repressed or protein unbound/bound states. We show that competition between switching timescale T t , polymer relaxation timescale τ p , and segmental relaxation timescale τ s can lead to non-trivial changes in chromatin organization, leading to changes in local compaction and contact probabilities. As a function of the switching timescale, the radius of gyration of chromatin shows a non-monotonic behavior with a prominent minimum when T t ≈ τ p and a maximum when T t ≈ τ s . We find that polymers with a small segment length exhibit a more compact structure than those with larger segment lengths. We also find that the switching can lead to higher contact probability and better mixing of far-away segments. Our study also shows that the nature of the distribution of chromatin clusters varies widely as we change the switching rate.

Liquid-liquid phase separation (LLPS) has emerged as a crucial biological phenomenon underlying the sequestration of macromolecules (such as proteins and nucleic acids) into membraneless organelles in cells. Unstructured and intrinsically disordered domains are known to facilitate multivalent interactions driving protein LLPS. We hypothesized that LLPS could be an intrinsic property of proteins/polypeptides but with distinct phase regimes irrespective of their sequence and structure. To examine this, we studied many (a total of 23) proteins/polypeptides with different structures and sequences for LLPS study in the presence and absence of molecular crowder, polyethylene glycol (PEG-8000). We showed that all proteins and even highly charged polypeptides (under study) can undergo liquid condensate formation, however with different phase regimes and intermolecular interactions. We further demonstrated that electrostatic, hydrophobic, and H-bonding or a combination of such intermolecular interactions plays a crucial role in individual protein/peptide LLPS., (© 2023. Springer Nature Limited.)

Simulating chromatin is crucial for predicting genome organization and dynamics. Although coarse-grained bead-spring polymer models are commonly used to describe chromatin, the relevant bead dimensions, elastic properties, and the nature of inter-bead potentials are unknown. Using nucleosome-resolution contact probability (Micro-C) data, we systematically coarse-grain chromatin and predict quantities essential for polymer representation of chromatin. We compute size distributions of chromatin beads for different coarse-graining scales, quantify fluctuations and distributions of bond lengths between neighboring regions, and derive effective spring constant values. Unlike the prevalent notion, our findings argue that coarse-grained chromatin beads must be considered as soft particles that can overlap, and we derive an effective inter-bead soft potential and quantify an overlap parameter. We also compute angle distributions giving insights into intrinsic folding and local bendability of chromatin. While the nucleosome-linker DNA bond angle naturally emerges from our work, we show two populations of local structural states. The bead sizes, bond lengths, and bond angles show different mean behavior at Topologically Associating Domain (TAD) boundaries and TAD interiors. We integrate our findings into a coarse-grained polymer model and provide quantitative estimates of all model parameters, which can serve as a foundational basis for all future coarse-grained chromatin simulations., (© 2023. The Author(s).)

Competing Interests: Declaration of interests The authors declare no competing interests.

Small Ubiquitin-like Modifier 1 (SUMO1) is an essential protein for many cellular functions, including regulation, signaling, etc., achieved by a process known as SUMOylation, which involves covalent attachment of SUMO1 to target proteins. SUMO1 also regulates the function of several proteins via non-covalent interactions involving the hydrophobic patch in the target protein identified as SUMO Binding or Interacting Motif (SBM/SIM). Here, we demonstrate a crucial functional potential of SUMO1 mediated by its non-covalent interactions with α-Synuclein, a protein responsible for many neurodegenerative diseases called α-Synucleinopathies. SUMO1 hinders the fibrillation of α-Synuclein, an intrinsically disordered protein (IDP) that undergoes a transition to β-structures during the fibrillation process. Using a plethora of biophysical techniques, we show that SUMO1 transiently binds to the N-terminus region of α-Synuclein non-covalently and causes structural compaction, which hinders the self-association process and thereby delays the fibrillation process. On the one hand, this study demonstrates an essential functional role of SUMO1 protein concerning neurodegeneration; it also illustrates the commonly stated mechanism that IDPs carry out multiple functions by structural adaptation to suit specific target proteins, on the other. Residue-level details about the SUMO1-α-Synuclein interaction obtained here also serve as a reliable approach for investigating the detailed mechanisms of IDP functions., (© 2023 The Protein Society.)

Chromatin attains its three-dimensional (3D) conformation by establishing contacts between different noncontiguous regions. Sterile Alpha Motif (SAM)-mediated polymerization of the polyhomeotic (PH) protein regulates subnuclear clustering of Polycomb Repressive Complex 1 (PRC1) and chromatin topology. The mutations that perturb the ability of the PH to polymerize, disrupt long-range chromatin contacts, alter Hox gene expression, and lead to developmental defects. To understand the underlying mechanism, we combined the experiments and theory to investigate the effect of this SAM domain mutation on nucleosome occupancy and accessibility on a genome wide scale. Our data show that disruption of PH polymerization because of SAM domain mutation decreases nucleosome occupancy and alters accessibility. Polymer simulations investigating the interplay between distant chromatin contacts and nucleosome occupancy, both of which are regulated by PH polymerization, suggest that nucleosome density increases when contacts between different regions of chromatin are established. Taken together, it appears that SAM domain-mediated PH polymerization biomechanically regulates the organization of chromatin at multiple scales from nucleosomes to chromosomes and we suggest that higher order organization can have a top-down causation effect on nucleosome occupancy., (© 2023 Amin et al.)

α-Synuclein (α-Syn) amyloids in synucleinopathies are suggested to be structurally and functionally diverse, reminiscent of prion-like strains. The mechanism of how the aggregation of the same precursor protein results in the formation of fibril polymorphs remains elusive. Here, we demonstrate the structure-function relationship of two polymorphs, pre-matured fibrils (PMFs) and helix-matured fibrils (HMFs), based on α-Syn aggregation intermediates. These polymorphs display the structural differences as demonstrated by solid-state NMR and mass spectrometry studies and also possess different cellular activities such as seeding, internalization, and cell-to-cell transfer of aggregates. HMFs, with a compact core structure, exhibit low seeding potency but readily internalize and transfer from one cell to another. The less structured PMFs lack transcellular transfer ability but induce abundant α-Syn pathology and trigger the formation of aggresomes in cells. Overall, the study highlights that the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Nucleosomes are the fundamental building blocks of chromatin that not only help in the folding of chromatin, but also in carrying epigenetic information. It is known that nucleosome sliding is responsible for dynamically organizing chromatin structure and the resulting gene regulation. Since sliding can move two neighboring nucleosomes physically close or away, can it play a role in the spreading of histone modifications? We investigate this by simulating a stochastic model that couples nucleosome dynamics with the kinetics of histone modifications. We show that the sliding of nucleosomes can affect the modification pattern as well as the time it takes to modify a given region of chromatin. Exploring different nucleosome densities and modification kinetic parameters, we show that nucleosome sliding can be important for creating histone modification domains. Our model predicts that nucleosome density coupled with sliding dynamics can create an asymmetric histone modification profile around regulatory regions. We also compute the probability distribution of modified nucleosomes and relaxation kinetics of modifications. Our predictions are comparable with known experimental results.

The size of amyloid seeds is known to modulate their autocatalytic amplification and cellular toxicity. However, the seed size-dependent secondary nucleation mechanism, toxicity, and disease-associated biological processes mediated by α-synuclein (α-Syn) fibrils are largely unknown. Using the cellular model and in vitro reconstitution, we showed that the size of α-Syn fibril seeds dictates not only their cellular internalization and associated cell death but also the distinct mechanisms of fibril amplification pathways involved in the pathological conformational change of α-Syn. Specifically, small fibril seeds showed elongation possibly through monomer addition at the fibril termini, whereas longer fibrils template the fibril amplification by surface-mediated nucleation as demonstrated by super-resolution microscopy. The distinct mechanism of fibril amplification and cellular uptake along with toxicity suggest that breakage of fibrils into seeds of different sizes determines the underlying pathological outcome of synucleinopathies.

Chromatin is known to be organized into multiple domains of varying sizes and compaction. While these domains are often imagined as static structures, they are highly dynamic and show cell-to-cell variability. Since processes such as gene regulation and DNA replication occur in the context of these domains, it is important to understand their organization, fluctuation, and dynamics. To simulate chromatin domains, one requires knowledge of interaction strengths among chromatin segments. Here, we derive interaction-strength parameters from experimentally known contact maps and use them to predict chromatin organization and dynamics. Taking two domains on the human chromosome as examples, we investigate its three-dimensional organization, size/shape fluctuations, and dynamics of different segments within a domain, accounting for hydrodynamic effects. Considering different cell types, we quantify changes in interaction strengths and chromatin shape fluctuations in different epigenetic states. Perturbing the interaction strengths systematically, we further investigate how epigenetic-like changes can alter the spatio-temporal nature of the domains. Our results show that heterogeneous weak interactions are crucial in determining the organization of the domains. Computing effective stiffness and relaxation times, we investigate how perturbations in interactions affect the solid- and liquid-like nature of chromatin domains. Quantifying dynamics of chromatin segments within a domain, we show how the competition between polymer entropy and interaction energy influence the timescales of loop formation and maintenance of stable loops., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Phase separation of biomolecules could be mediated by both specific and non-specific interactions. How the interplay between non-specific and specific interactions along with polymer entropy influences phase separation is an open question. We address this question by simulating self-associating molecules as polymer chains with a short core stretch that forms the specifically interacting functional interface and longer non-core regions that participate in non-specific/promiscuous interactions. Our results show that the interplay of specific (strength, ϵsp) and non-specific interactions (strength, ϵns) could result in phase separation of polymers and its transition to solid-like aggregates (mature state). In the absence of ϵns, the polymer chains do not dwell long enough in the vicinity of each other to undergo phase separation and transition into a mature state. On the other hand, in the limit of strong ϵns, the assemblies cannot transition into the mature state and form a non-specific assembly, suggesting an optimal range of interactions favoring mature multimers. In the scenario where only a fraction (Nfrac) of the non-core regions participate in attractive interactions, we find that slight modifications to either ϵns or Nfrac can result in dramatically altered self-assembled states. Using a combination of heterogeneous and homogeneous mix of polymers, we establish how this interplay between interaction energies dictates the propensity of biomolecules to find the correct binding partner at dilute concentrations in crowded environments., Competing Interests: The authors have declared that no competing interests exist.

During cell devision, maintaining the epigenetic information encoded in histone modification patterns is crucial for survival and identity of cells. The faithful inheritance of the histone marks from the parental to the daughter strands is a puzzle, given that each strand gets only half of the parental nucleosomes. Mapping DNA replication and reconstruction of modifications to equivalent problems in communication of information, we ask how well enzymes can recover the parental modifications, if they were ideal computing machines. Studying a parameter regime where realistic enzymes can function, our analysis predicts that enzymes may implement a critical threshold filling algorithm which fills unmodified regions of length at most k. This algorithm, motivated from communication theory, is derived from the maximum à posteriori probability (MAP) decoding which identifies the most probable modification sequence based on available observations. Simulations using our method produce modification patterns similar to what has been observed in recent experiments. We also show that our results can be naturally extended to explain inheritance of spatially distinct antagonistic modifications., Competing Interests: The authors have declared that no competing interests exist.

Understanding kinetic control of biological processes is as important as identifying components that constitute pathways. Insulin signaling is central for almost all metazoans, and its perturbations are associated with various developmental disorders, metabolic diseases, and aging. While temporal phosphorylation changes and kinetic constants have provided some insights, constant or variable parameters that establish and maintain signal topology are poorly understood. Here, we report kinetic parameters that encode insulin concentration and nutrient-dependent flow of information using iterative experimental and mathematical simulation-based approaches. Our results illustrate how dynamics of distinct phosphorylation events collectively contribute to selective kinetic gating of signals and maximum connectivity of the signaling cascade under normo-insulinemic but not hyper-insulinemic states. In addition to identifying parameters that provide predictive value for maintaining the balance between metabolic and growth-factor arms, we posit a kinetic basis for the emergence of insulin resistance. Given that pulsatile insulin secretion during a fasted state precedes a fed response, our findings reveal rewiring of insulin signaling akin to memory and anticipation, which was hitherto unknown. Striking disparate temporal behavior of key phosphorylation events that destroy the topology under hyper-insulinemic states underscores the importance of unraveling regulatory components that act as bandwidth filters. In conclusion, besides providing fundamental insights, our study will help in identifying therapeutic strategies that conserve coupling between metabolic and growth-factor arms, which is lost in diseases and conditions of hyper-insulinemia., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The establishment of centromeric chromatin and its propagation by the centromere-specific histone CENPA is mediated by epigenetic mechanisms in most eukaryotes. DNA replication origins, origin binding proteins, and replication timing of centromere DNA are important determinants of centromere function. The epigenetically regulated regional centromeres in the budding yeast Candida albicans have unique DNA sequences that replicate earliest in every chromosome and are clustered throughout the cell cycle. In this study, the genome-wide occupancy of the replication initiation protein Orc4 reveals its abundance at all centromeres in C. albicans Orc4 is associated with four different DNA sequence motifs, one of which coincides with tRNA genes (tDNA) that replicate early and cluster together in space. Hi-C combined with genome-wide replication timing analyses identify that early replicating Orc4-bound regions interact with themselves stronger than with late replicating Orc4-bound regions. We simulate a polymer model of chromosomes of C. albicans and propose that the early replicating and highly enriched Orc4-bound sites preferentially localize around the clustered kinetochores. We also observe that Orc4 is constitutively localized to centromeres, and both Orc4 and the helicase Mcm2 are essential for cell viability and CENPA stability in C. albicans Finally, we show that new molecules of CENPA are recruited to centromeres during late anaphase/telophase, which coincides with the stage at which the CENPA-specific chaperone Scm3 localizes to the kinetochore. We propose that the spatiotemporal localization of Orc4 within the nucleus, in collaboration with Mcm2 and Scm3, maintains centromeric chromatin stability and CENPA recruitment in C. albicans ., (© 2021 Sreekumar et al.; Published by Cold Spring Harbor Laboratory Press.)

An important question in the context of the three-dimensional organization of chromosomes is the mechanism of formation of large loops between distant basepairs. Recent experiments suggest that the formation of loops might be mediated by loop extrusion factor proteins such as cohesin. Experiments on cohesin have shown that cohesins walk diffusively on the DNA and that nucleosomes act as obstacles to the diffusion, lowering the permeability and hence reducing the effective diffusion constant. An estimation of the times required to form the loops of typical sizes seen in Hi-C experiments using these low-effective-diffusion constants leads to times that are unphysically large. The puzzle then is the following: how does a cohesin molecule diffusing on the DNA backbone achieve speeds necessary to form the large loops seen in experiments? We propose a simple answer to this puzzle and show that although at low densities, nucleosomes act as barriers to cohesin diffusion, beyond a certain concentration they can reduce loop formation times because of a subtle interplay between the nucleosome size and the mean linker length. This effect is further enhanced on considering stochastic binding kinetics of nucleosomes on the DNA backbone and leads to predictions of lower loop formation times than might be expected from a naive obstacle picture of nucleosomes., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Amyloid fibrils are typically associated with neurodegenerative diseases. Recent studies have suggested that, similar to prions, many amyloid proteins are infectious in nature and may cause spreading and dissemination of diseases. Typical amyloid infection propagates by recruiting functional proteins into amyloidogenic form and multiplying by breaking the existing fibril. In this study, we model the kinetics of fibril growth through breakage and the subsequent elongation process, similar to the prion infection process. Using kinetic Monte Carlo simulations as well as mathematical counting methods, we show how the measurable quantities like the 50% aggregation time ( T 50 ) and the maximum growth rate ( V max ) scale with various parameters in the problem. This study has a direct application where it can be used to understand experiments that amplify the minute amount of amyloid seeds present in biological fluid for early detection of human disease. Using the knowledge from our simulations, we can predict the initial seed concentration, known as the filament kinetics.

In our cells, DNA is folded and packed with the help of many proteins into chromatin whose basic unit is a nucleosome-DNA wrapped around octamer of histone proteins. The chain of nucleosomes is further folded and arranged into many layers and has a dynamic organization. How does the complex chromatin organization emerge from interactions among DNA, histones, and non-histone proteins have been a question of great interest. Here we review recent literature that investigated how nucleosome positioning and nucleosome-mediated interactions drive chromatin organization. Unlike our earlier understanding, chromatin is organized into 3D domains of various sizes having irregularly organized nucleosomes. These domains emerge due to heterogeneous nucleosome positioning and diverse inter-nucleosome interactions that vary in space and time., (Copyright © 2020. Published by Elsevier Ltd.)

α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson's disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid-liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson's disease mutations, also promote α-Syn liquid-liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson's disease pathogenesis.

Microtubules (MTs) are bio-polymers, composed of tubulin proteins, involved in several functions such as cell division, transport of cargoes within cells, maintaining cellular structures etc. Their kinetics are often affected by chemical modifications on the filament known as Post Translational Modifications (PTMs). Acetylation is a PTM which occurs on the luminal surface of the MT lattice and has been observed to reduce the lateral interaction between tubulins on adjacent protofilaments. Depending on the properties of the acetylase enzyme αTAT1 and the structural features of MTs, the patterns of acetylation formed on MTs are observed to be quite diverse. In this study, we present a multi-protofilament model with spatially heterogeneous patterns of acetylation, and investigate how the local kinetic differences arising from heterogeneity affect the global kinetics of MT filaments. From the computational study we conclude that a filament with spatially uniform acetylation is least stable against disassembly, while ones with more clustered acetylation patterns may provide better resistance against disassembly. The increase in disassembly times for clustered pattern as compared to uniform pattern can be up to fifty percent for identical amounts of acetylation. Given that acetylated MTs affect several cellular functions as well as diseases such as cancer, our study indicates that spatial patterns of acetylation need to be focused on, apart from the overall amount of acetylation.

How chromatin is folded on the length scale of a gene is an open question. Recent experiments have suggested that, in vivo, chromatin is folded in an irregular manner and not as an ordered fiber with a width of 30 nm that is expected from theories of higher order packaging. Using computational methods, we examine how the interplay between DNA-bending nonhistone proteins, histone tails, intrachromatin electrostatic, and other interactions decide the nature of the packaging of chromatin. We show that although the DNA-bending nonhistone proteins make the chromatin irregular, they may not alter the packing density and size of the fiber. We find that the length of the interacting region and intrachromatin electrostatic interactions influence the packing density, clustering of nucleosomes, and the width of the chromatin fiber. Our results suggest that the heterogeneity in the interaction pattern will play an important role in deciding the nature of the packaging of chromatin., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We present a physics-based polymer model that can investigate 3D organization of chromatin accounting for DNA elasticity, DNA-bending due to nucleosomes, and 1D organization of nucleosomes along DNA. We find that the packing density of chromatin oscillates between densities corresponding to highly folded and extended configurations as we change the nucleosome organization (length of linker DNA). We compute the looping probability of chromatin and show that the presence of nucleosomes increases the looping probability of the chain compared to that of a bare DNA. We also show that looping probability has a large variability depending on the nature of nucleosome organization and density of linker histones.

Positioning of nucleosomes along the genomic DNA is crucial for many cellular processes that include gene regulation and higher order packaging of chromatin. The question of how nucleosome-positioning information from a parent chromatin gets transferred to the daughter chromatin is highly intriguing. Accounting for experimentally known coupling between replisome movement and nucleosome dynamics, we propose a model that can obtain de novo nucleosome assembly similar to what is observed in recent experiments. Simulating nucleosome dynamics during replication, we argue that short pausing of the replication fork, associated with nucleosome disassembly, can be a event crucial for communicating nucleosome positioning information from parent to daughter. We show that the interplay of timescales between nucleosome disassembly (τp) at the replication fork and nucleosome sliding behind the fork (τs) can give rise to a rich 'phase diagram' having different inherited patterns of nucleosome organization. Our model predicts that only when τp ≥ τs the daughter chromatin can inherit nucleosome positioning of the parent.

Aggregation of α-synuclein (α-Syn) into neurotoxic oligomers and amyloid fibrils is suggested to be the pathogenic mechanism for Parkinson's disease (PD). Recent studies have indicated that oligomeric species of α-Syn are more cytotoxic than their mature fibrillar counterparts, which are responsible for dopaminergic neuronal cell death in PD. Therefore, the effective therapeutic strategies for tackling aggregation-associated diseases would be either to prevent aggregation or to modulate the aggregation process to minimize the formation of toxic oligomers during aggregation. In this work, we showed that arginine-substituted α-Syn ligands, based on the most aggregation-prone sequence of α-Syn, accelerate the protein aggregation in a concentration-dependent manner. To elucidate the mechanism by which Arg-substituted peptides could modulate α-Syn aggregation kinetics, we performed surface plasmon resonance (SPR) spectroscopy, nuclear magnetic resonance (NMR) studies, and all-atom molecular dynamics (MD) simulation. The SPR analysis showed a high binding potency of these peptides with α-Syn but one that was nonspecific in nature. The two-dimensional NMR studies suggest that a large stretch within the C-terminus of α-Syn displays a chemical shift perturbation upon interacting with Arg-substituted peptides, indicating C-terminal residues of α-Syn might be responsible for this class of peptide binding. This is further supported by MD simulation studies in which the Arg-substituted peptide showed the strongest interaction with the C-terminus of α-Syn. Overall, our results suggest that the binding of Arg-substituted ligands to the highly acidic C-terminus of α-Syn leads to reduced charge density and flexibility, resulting in accelerated aggregation kinetics. This may be a potentially useful strategy while designing peptides, which act as α-Syn aggregation modulators.

Characterising complex kinetics of non-equilibrium self-assembly of bio-filaments is of general interest. Dynamic instability in microtubules, consisting of successive catastrophes and rescues, is observed to occur as a result of the non-equilibrium conversion of GTP-tubulin to GDP-tubulin. We study this phenomenon using a model for microtubule kinetics with GTP/GDP state-dependent polymerisation, depolymerisation and hydrolysis of subunits. Our results reveal a sharp switch-like transition in the mean velocity of the filaments, from a growth phase to a shrinkage phase, with an associated co-existence of the two phases. This transition is reminiscent of the discontinuous phase transition across the liquid-gas boundary. We probe the extent of discontinuity in the transition quantitatively using characteristic signatures such as bimodality in velocity distribution, variance and Binder cumulant, and also hysteresis behaviour of the system. We further investigate ageing behaviour in catastrophes of the filament, and find that the multi-step nature of catastrophes is intensified in the vicinity of the switching transition. This assumes importance in the context of Microtubule Associated Proteins which have the potential of altering kinetic parameter values.

Molecular motors and cytoskeletal filaments work collectively most of the time under opposing forces. This opposing force may be due to cargo carried by motors or resistance coming from the cell membrane pressing against the cytoskeletal filaments. Some recent studies have shown that the collective maximum force (stall force) generated by multiple cytoskeletal filaments or molecular motors may not always be just a simple sum of the stall forces of the individual filaments or motors. To understand this excess or deficit in the collective force, we study a broad class of models of both cytoskeletal filaments and molecular motors. We argue that the stall force generated by a group of filaments or motors is additive, that is, the stall force of N number of filaments (motors) is N times the stall force of one filament (motor), when the system is reversible at stall. Conversely, we show that this additive property typically does not hold true when the system is irreversible at stall. We thus present a novel and unified understanding of the existing models exhibiting such non-addivity, and generalise our arguments by developing new models that demonstrate this phenomena. We also propose a quantity similar to thermodynamic efficiency to easily predict this deviation from stall-force additivity for filament and motor collectives.

Why most of the in vivo experiments do not find the 30-nm chromatin fiber, well studied in vitro, is a puzzle. Two basic physical inputs that are crucial for understanding the structure of the 30-nm fiber are the stiffness of the linker DNA and the relative orientations of the DNA entering/exiting nucleosomes. Based on these inputs we simulate chromatin structure and show that the presence of non-histone proteins, which bind and locally bend linker DNA, destroys any regular higher order structures (e.g., zig-zag). Accounting for the bending geometry of proteins like nhp6 and HMG-B, our theory predicts phase-diagram for the chromatin structure as a function of DNA-bending non-histone protein density and mean linker DNA length. For a wide range of linker lengths, we show that as we vary one parameter, that is, the fraction of bent linker region due to non-histone proteins, the steady-state structure will show a transition from zig-zag to an irregular structure-a structure that is reminiscent of what is observed in experiments recently. Our theory can explain the recent in vivo observation of irregular chromatin having co-existence of finite fraction of the next-neighbor (i + 2) and neighbor (i + 1) nucleosome interactions., Competing Interests: The authors have declared that no competing interests exist.

Gal3p is an allosteric monomeric protein that activates the GAL genetic switch of Saccharomyces cerevisiae in response to galactose. Expression of constitutive mutant of Gal3p or overexpression of wild-type Gal3p activates the GAL switch in the absence of galactose. These data suggest that Gal3p exists as an ensemble of active and inactive conformations. Structural data have indicated that Gal3p exists in open (inactive) and closed (active) conformations. However, a mutant of Gal3p that predominantly exists in inactive conformation and is yet capable of responding to galactose has not been isolated. To understand the mechanism of allosteric transition, we have isolated a triple mutant of Gal3p with V273I, T404A, and N450D substitutions, which, upon overexpression, fails to activate the GAL switch on its own but activates the switch in response to galactose. Overexpression of Gal3p mutants with single or double mutations in any of the three combinations failed to exhibit the behavior of the triple mutant. Molecular dynamics analysis of the wild-type and the triple mutant along with two previously reported constitutive mutants suggests that the wild-type Gal3p may also exist in super-open conformation. Furthermore, our results suggest that the dynamics of residue F237 situated in the hydrophobic pocket located in the hinge region drives the transition between different conformations. Based on this study, we suggest that conformational selection mechanism is the driving force in the allosteric transition of Gal3p, which may have implications in other signaling pathways involving monomeric proteins., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Self-assembly of proteins into ordered, fibrillar structures is a commonly observed theme in biology. It has been observed that diverse set of proteins (e.g., alpha-synuclein, insulin, TATA-box binding protein, Sup35, p53), independent of their sequence, native structure, or function could self-assemble into highly ordered structures known as amyloids. What are the crucial features underlying amyloidogenesis that make it so generic? Using coarse-grained simulations of peptide self-assembly, we argue that variation in two physical parameters-bending stiffness of the polypeptide and strength of intermolecular interactions-can give rise to many of the structural features typically associated with amyloid self-assembly. We show that the interplay between these two factors gives rise to a rich phase diagram displaying high diversity in aggregated states. For certain parameters, we find a bimodal distribution for the order parameter implying the coexistence of ordered and disordered aggregates. Our findings may explain the experimentally observed variability including the "off-pathway" aggregated structures. Further, we demonstrate that sequence-dependence and protein-specific signatures could be mapped to our coarse-grained framework to study self-assembly behavior of realistic systems such as the STVIIE peptide and Aβ42. The work also provides certain guiding principles that could be used to design novel peptides with desired self-assembly properties, by tuning a few physical parameters.

It is being increasingly realized that nucleosome organization on DNA crucially regulates DNA-protein interactions and the resulting gene expression. While the spatial character of the nucleosome positioning on DNA has been experimentally and theoretically studied extensively, the temporal character is poorly understood. Accounting for ATPase activity and DNA-sequence effects on nucleosome kinetics, we develop a theoretical method to estimate the time of continuous exposure of binding sites of non-histone proteins (e.g. transcription factors and TATA binding proteins) along any genome. Applying the method to Saccharomyces cerevisiae, we show that the exposure timescales are determined by cooperative dynamics of multiple nucleosomes, and their behavior is often different from expectations based on static nucleosome occupancy. Examining exposure times in the promoters of GAL1 and PHO5, we show that our theoretical predictions are consistent with known experiments. We apply our method genome-wide and discover huge gene-to-gene variability of mean exposure times of TATA boxes and patches adjacent to TSS (+1 nucleosome region); the resulting timescale distributions have non-exponential tails., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Amyloid formation is associated with various pathophysiological conditions like Alzheimer's and Parkinson's diseases as well as many useful functions. The hallmark of amyloid assemblies is a conformational transition of the constituent proteins into a β - sheet rich filament. Accounting for this conformational transition in amyloidogenic proteins, we develop an analytically solvable model that can probe the dynamics of an ensemble of single filaments. Using the theory and Monte Carlo simulations, we show the presence of two kinetic regimes for the growth of a self-assembling filament - switching-dependent and -independent growth regimes. We observe a saturation in fibril elongation velocities at higher concentrations in the first regime, providing a novel explanation to the concentration-independence of growth velocities observed experimentally. We also compute the length fluctuation of the filaments to characterize aggregate heterogeneity. From the early velocities and length fluctuation, we propose a novel way of estimating the conformational switching rate. Our theory predicts a kinetic phase diagram that has three distinct phases - short oligomers/monomers, disordered aggregates and β-rich filaments. The model also predicts the force generation potential and the intermittent growth of amyloid fibrils evident from single molecular experiments. Our model could contribute significantly to the physical understanding of amyloid aggregation.

Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcription factors (TFs) and other non-histone proteins accessing genomic DNA. How the binding competition between nucleosomes and TFs leads to transcriptionally compatible promoter states is an open question. Here, we present a computational study of the nucleosome dynamics and organization in the promoter region of PHO5 gene in Saccharomyces cerevisiae. Introducing a model for nucleosome kinetics that takes into account ATP-dependent remodeling activity, DNA sequence effects, and kinetics of TFs (Pho4p), we compute the probability of obtaining different "promoter states" having different nucleosome configurations. Comparing our results with experimental data, we argue that the presence of local remodeling activity (LRA) as opposed to basal remodeling activity (BRA) is crucial in determining transcriptionally active promoter states. By modulating the LRA and Pho4p binding rate, we obtain different mRNA distributions-Poisson, bimodal, and long-tail. Through this work we explain many features of the PHO5 promoter such as sequence-dependent TF accessibility and the role of correlated dynamics between nucleosomes and TFs in opening/coverage of the TATA box. We also obtain possible ranges for TF binding rates and the magnitude of LRA.

Microtubules are nano-machines that grow and shrink stochastically, making use of the coupling between chemical kinetics and mechanics of its constituent protofilaments (PFs). We investigate the stability and shrinkage of microtubules taking into account inter-protofilament interactions and bending interactions of intrinsically curved PFs. Computing the free energy as a function of PF tip position, we show that the competition between curvature energy, inter-PF interaction energy and entropy leads to a rich landscape with a series of minima that repeat over a length-scale determined by the intrinsic curvature. Computing Langevin dynamics of the tip through the landscape and accounting for depolymerization, we calculate the average unzippering and shrinkage velocities of GDP protofilaments and compare them with the experimentally known results. Our analysis predicts that the strength of the inter-PF interaction (E(s)(m)) has to be comparable to the strength of the curvature energy (E(b)(m)) such that E(s)(m) - E(b)(m) ≈ 1kBT, and questions the prevalent notion that unzippering results from the domination of bending energy of curved GDP PFs. Our work demonstrates how the shape of the free energy landscape is crucial in explaining the mechanism of MT shrinkage where the unzippered PFs will fluctuate in a set of partially peeled off states and subunit dissociation will reduce the length.

How cytoskeletal filaments collectively undergo growth and shrinkage is an intriguing question. Collective properties of multiple bio-filaments (actin or microtubules) undergoing hydrolysis have not been studied extensively earlier within simple theoretical frameworks. In this paper, we study the collective dynamical properties of multiple filaments under force, and demonstrate the distinct properties of a multi-filament system in comparison to a single filament. Comparing stochastic simulation results with recent experimental data, we show that multi-filament collective catastrophes are slower than catastrophes of single filaments. Our study also shows further distinctions as follows: (i) force-dependence of the cap-size distribution of multiple filaments are quantitatively different from that of single filaments, (ii) the diffusion constant associated with the system length fluctuations is distinct for multiple filaments, and (iii) switching dynamics of multiple filaments between capped and uncapped states and the fluctuations therein are also distinct. We build a unified picture by establishing interconnections among all these collective phenomena. Additionally, we show that the collapse times during catastrophes can be sharp indicators of collective stall forces exceeding the additive contributions of single filaments.

We investigate collective dynamics of branched actin networks growing against a rigid movable wall constrained by a resistive force. Computing the force velocity relations, we show that the stall force of such networks depends not only on the average number of filaments touching the wall, but also on the amount of fluctuation of the leading edge of the network. These differences arise due to differences in the network architecture, namely, distance between two adjacent branching points and the initial distance of the starting filament from the wall, with their relative magnitudes influencing the nature of the force velocity curves (convex versus concave). We also show that the introduction of branching results in nonmonotonic diffusion constant, a quantity that measures the growth in length fluctuation of the leading edge of the network, as a function of externally applied force. Together our results demonstrate how the collective dynamics of a branched network differs from that of a parallel filament network.

Protein aggregation and amyloid formation are known to play a role both in diseases and in biological functions. Transcription factor p53 plays a major role in tumor suppression by maintaining genomic stability. Recent studies have suggested that amyloid formation of p53 could lead to its loss of physiological function as a tumor suppressor. Here, we investigated the intrinsic amyloidogenic nature of wild-type p53 using sequence analysis. We used bioinformatics and aggregation prediction algorithms to establish the evolutionarily conserved nature of aggregation-prone sequences in wild-type p53. Further, we analyzed the amyloid forming capacity of conserved and aggregation-prone p53-derived peptides PILTIITL and YFTLQI in vitro using various biophysical techniques, including all atom molecular dynamics simulation. Finally, we probed the seeding ability of the PILTIITL peptide on p53 aggregation in vitro and in cells. Our data demonstrate the intrinsic amyloid forming ability of a sequence stretch of the p53 DNA binding domain (DBD) and its aggregation templating behavior on full-length and p53 core domain. Therefore, p53 aggregation, instigated through an amyloidogenic segment in its DBD, could be a putative driving force for p53 aggregation in vivo.

Physical properties of the extracellular matrix (ECM) are known to regulate cellular processes ranging from spreading to differentiation, with alterations in cell phenotype closely associated with changes in physical properties of cells themselves. When plated on substrates of varying stiffness, fibroblasts have been shown to exhibit stiffness matching property, wherein cell cortical stiffness increases in proportion to substrate stiffness up to 5 kPa, and subsequently saturates. Similar mechanoadaptation responses have also been observed in other cell types. Trypsin de-adhesion represents a simple experimental framework for probing the contractile mechanics of adherent cells, with de-adhesion timescales shown to scale inversely with cortical stiffness values. In this study, we combine experiments and computation in deciphering the influence of substrate properties in regulating de-adhesion dynamics of adherent cells. We first show that NIH 3T3 fibroblasts cultured on collagen-coated polyacrylamide hydrogels de-adhere faster on stiffer substrates. Using a simple computational model, we qualitatively show how substrate stiffness and cell-substrate bond breakage rate collectively influence de-adhesion timescales, and also obtain analytical expressions of de-adhesion timescales in certain regimes of the parameter space. Finally, by comparing stiffness-dependent experimental and computational de-adhesion responses, we show that faster de-adhesion on stiffer substrates arises due to force-dependent breakage of cell-matrix adhesions. In addition to illustrating the utility of employing trypsin de-adhesion as a biophysical tool for probing mechanoadaptation, our computational results highlight the collective interplay of substrate properties and bond breakage rate in setting de-adhesion timescales.

We investigate how DNA sequence, ATP-dependent chromatin remodeling and nucleosome-depleted 'barriers' co-operate to determine the kinetics of nucleosome organization, in a stochastic model of nucleosome positioning and dynamics. We find that 'statistical' positioning of nucleosomes against 'barriers', hypothesized to control chromatin structure near transcription start sites, requires active remodeling and therefore cannot be described using equilibrium statistical mechanics. We show that, unlike steady-state occupancy, DNA site exposure kinetics near a barrier is dominated by DNA sequence rather than by proximity to the barrier itself. The timescale for formation of positioning patterns near barriers is proportional to the timescale for active nucleosome eviction. We also show that there are strong gene-to-gene variations in nucleosome positioning near barriers, which are eliminated by averaging over many genes. Our results suggest that measurement of nucleosome kinetics can reveal information about sequence-dependent regulation that is not apparent in steady-state nucleosome occupancy.

Glycosaminoglycans (GAGs) have been reported to play a significant role in amyloid formation of a wide range of proteins/peptides either associated with diseases or native biological functions. The exact mechanism by which GAGs influence amyloid formation is not clearly understood. Here, we studied two closely related peptides, glucagon-like peptide 1 (GLP1) and glucagon-like peptide 2 (GLP2), for their amyloid formation in the presence and absence of the representative GAG heparin using various biophysical and computational approaches. We show that the aggregation and amyloid formation by these peptides follow distinct mechanisms: GLP1 follows nucleation-dependent aggregation, whereas GLP2 forms amyloids without any significant lag time. Investigating the role of heparin, we also found that heparin interacts with GLP1, accelerates its aggregation, and gets incorporated within its amyloid fibrils. In contrast, heparin neither affects the aggregation kinetics of GLP2 nor gets embedded within its fibrils. Furthermore, we found that heparin preferentially influences the stability of the GLP1 fibrils over GLP2 fibrils. To understand the specific nature of the interaction of heparin with GLP1 and GLP2, we performed all-atom MD simulations. Our in silico results show that the basic-nonbasic-basic (B-X-B) motif of GLP1 (K28-G29-R30) facilitates the interaction between heparin and peptide monomers. However, the absence of such a motif in GLP2 could be the reason for a significantly lower strength of interaction between GLP2 and heparin. Our study not only helps to understand the role of heparin in inducing protein aggregation but also provides insight into the nature of heparin-protein interaction.

Microtubules consist of 13 protofilaments arranged in the form of a cylinder. The protofilaments are composed of longitudinally attached tubulin dimers that can exist in either a less curved state [GTP-bound tubulin (T)] or a more curved state [GDP-bound tubulin (D)]. Hydrolysis of T into D leaves the straight and laterally attached protofilaments of the microtubule in a mechanically stressed state, thus leading to their unzipping. The elastic energy in the unzipping protofilaments can be harnessed by a force transducer such as the Dam1-kinetochore ring complex in order to exert pulling force on chromosomes during cell division. In the present paper we develop a simple continuum model to obtain this pulling force as a function of the mechanical properties of protofilaments and the size of the Dam1-kinetochore ring. We also extend this model to investigate the role played by the T subunits found at the plus end of the microtubule (the T cap) on the mechanical stability of microtubules.

Recent measurements of the distribution of end-to-end distance in short DNA molecules infer cooperative stretching fluctuations. The assumptions underlying the analysis can be questioned if transient, thermally induced defects producing a localized decrease in bending stiffness are present in thermal equilibrium, such as regions in which DNA melts locally (bubbles), sustains large-angle bends (kinks), or can locally transform into an alternative (S-DNA) state. We study a generalized discrete worm-like chain model for DNA, capable of describing these experiments, showing that the model yields accurate fits to available experimental data. Our results indicate that DNA bending arising from such localized defects, rather than solely stretching, can be an equal contributor to end-to-end distance fluctuations for 35-bp DNA and contributes nontrivially to such fluctuations at all scales below the persistence length. The analysis suggests that such fluctuations should exhibit a scale-dependent cooperativity, specifically relevant in determining the behavior of short chains, but which saturates rapidly to a length-independent value for longer DNA, to ensure a consistent physical description of DNA across multiple scales. Our approach provides a minimal, yet accurate, coarse-grained description of DNA at the subpersistence length scales of current experimental interest., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Depolymerizing cytoskeletal filaments are involved in cell division, cell motility, and other cellular functions. Understanding the dynamics of depolymerization is as important as understanding the dynamics of polymerization. We study nonequilibrium depolymerization of actin filaments using a simple two-state model. We show that the polymerization history influences the dynamics of depolymerization as well as the length fluctuations during depolymerization. We also simulate depolymerization under different experimentally feasible conditions. Under conditions of constant concentration, we show that the depolymerization happens in two regimes. Under the conditions of mass conservation, the depolymerization can have three regimes.

Uncovering mechanisms that control the dynamics of microtubules is fundamental for our understanding of multiple cellular processes such as chromosome separation and cell motility. Building on previous theoretical work on the dynamic instability of microtubules, we propose here a stochastic model that includes all relevant biochemical processes that affect the dynamics of microtubule plus-end, namely, the binding of GTP-bound monomers, unbinding of GTP- and GDP-bound monomers, and hydrolysis of GTP monomers. The inclusion of dissociation processes, present in our approach but absent from many previous studies, is essential to guarantee the thermodynamic consistency of the model. Our theoretical method allows us to compute all dynamic properties of microtubules explicitly. Using experimentally determined rates, it is found that the cap size is ∼3.6 layers, an estimate that is compatible with several experimental observations. In the end, our model provides a comprehensive description of the dynamic instability of microtubules that includes not only the statistics of catastrophes but also the statistics of rescues., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)