Disparity in anomalous diffusion of proteins searching for their target DNA sites in a crowded medium is controlled by the size, shape and mobility of macromolecular crowders

Dey, Pinki; Bhattacherjee, Arnab

doi:10.1039/c8sm01933a

Cited by 24 publications

(36 citation statements)

References 59 publications

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“…Further systematic investigation of diffusion in crowded media (22,37) supported the viewpoint and predicted that the role of bulk crowders is to prevent the dissociation of DBPs from the DNA surface and thereby promoting 1D diffusion of the searching protein along the DNA contour over 3D diffusion (38,39). In contrast, previous studies indicate that purely repulsive crowders play a role to decouple the searching protein from the crowding agents during the 1D search regime on a linear DNA stretch (40,41). The observation is along the line of Asakura-Oosawa model (AOM) (42) of crowder action, where the crowding agents, due to excluded volume interactions with the DNA molecule result in a preferentially depleted volume around the DNA interface.…”

Section: Introductionmentioning

confidence: 87%

“…In the presence of bulk crowders, the protein moves mainly in two ways: either it diffuses three-dimensionally away from the DNA molecule (see Fig 1A) or one-dimensionally close to the DNA surface. During 3D diffusion, the protein interacts with the randomly moving crowder molecules and its overall diffusivity is governed by the concentration, size, shape, and mobility of the crowding agents (41). In comparison, protein diffusion during the 1D search regime was found to be significantly different in the presence of purely repulsive crowders (40,41).…”

Section: Bulk Crowding Modulates Environment For 1d and 3d Diffusion mentioning

confidence: 99%

“…Our objective is to investigate if the similar kind of crowding environment, but with nonspecific crowder affinity that PEG also features in contradiction to its believed 'inert' character, enhances the facilitated diffusion of DBPs, and if yes, to probe the underlying molecular mechanism. To this end, one should note that the present crowder model is not intended to mimic the other features of cytoplasmic crowders such as size heterogeneity that plays crucial roles in regulating various dynamic phenomena (41,58,59), rather to investigate how attractive interactions between searching protein and the crowder molecules affect the diffusion of the former. While a crowder model that mimics all the features of cytoplasmic crowders including their compressibility, malleability, size to charge ratio etc.…”

Section: Crowder Modelmentioning

confidence: 99%

“…The newly formed depletion zone is referred as composite depletion zone that features a narrower width because of attractive depletion force. Schematic description of the formation of composite depletion zone is depicted in Fig S2. To identify various search modes adopted by the diffusing protein, we follow the description similar to our previous works (40,41). Briefly, the protein is said to perform sliding if at least 70% of the recognition helix of Sap-1 is within the DNA major groove with an orientation angle of < 25 • , and the center of mass of the recognition helix lies within 8 Å to the closest DNA base pair in the absence of crowder molecules (see Fig S3).…”

Section: Estimation Of Depletion Layer Dimension and Characterizing Smentioning

confidence: 99%

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Structural Basis of Enhanced Facilitated Diffusion of DNA Binding Proteins in Crowded Cellular Milieu

Bhattarcharjee

Dey

2019

Preprint

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DNA binding proteins (DBPs) rapidly recognize and specifically associate with their target DNA sites inside cell nucleus that contains up to 400 g/L macromolecules, most of which are proteins. While the fast association between DBPs and DNA is explained by a facilitated diffusion mechanism, where DBPs adopt a weighted combination of 3D diffusion and 1D sliding and hopping modes of transportation, the role of cellular environment that contains many nonspecifically interacting proteins and other biomolecules is mostly overlooked. By performing large scale computational simulations with an appropriately tuned model of protein and DNA in the presence of nonspecifically interacting bulk and DNA bound crowders (genomic crowders), we demonstrate the structural basis of the enhanced facilitated diffusion of DBPs inside a crowded cellular milieu through novel 1D scanning mechanisms. In the presence of bulk crowders, we identify the protein to float along the DNA under the influence of protein-crowder nonspecific interactions. The search mode is distinctly different compared to usual 1D sliding and hopping dynamics where protein diffusion is regulated by the DNA electrostatics. In contrast, the presence of genomic crowders expedite the target search process by transporting the protein over DNA segments through the formation of a transient protein-crowder bridged complex. By analyzing the ruggedness of the associated potential energy landscape, we underpin the molecular origin of the kinetic advantages of these search modes and show that they successfully explain the experimentally observed acceleration of facilitated diffusion of DBPs by molecular crowding agents and crowder concentration dependent enzymatic activity of transcription factors. Our findings provide crucial insights into gene regulation kinetics inside the crowded cellular milieu.SIGNIFICANCE 10-40% of the intracellular volume is occupied by proteins, and other biomolecules, collectively known as macromolecular crowders. Their presence has been found to promote faster translocation of DNA binding proteins (DBPs) during the search of their target DNA sites for crucial cellular processes. Using molecular simulations, we probe the underlying structural basis and underscore the existence of novel DNA scanning mechanisms actuated by protein-crowder nonspecific interactions. We show that the observed search modes are kinetically beneficial and can successfully explain the acceleration of facilitated diffusion of DBPs by molecular crowding agents and crowderconcentration dependent enzymatic activity of transcription factors.Our study sheds new light on the long-standing facilitated diffusion problem of DBPs in the crowded cellular environment for regulating gene expression.

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Section: Introductionmentioning

confidence: 87%

Section: Bulk Crowding Modulates Environment For 1d and 3d Diffusion mentioning

confidence: 99%

Section: Crowder Modelmentioning

confidence: 99%

Section: Estimation Of Depletion Layer Dimension and Characterizing Smentioning

confidence: 99%

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Structural Basis of Enhanced Facilitated Diffusion of DNA Binding Proteins in Crowded Cellular Milieu

Bhattarcharjee

Dey

2019

Preprint

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“…We adopted a coarse-grained representation of protein, where each amino acid is represented by a single bead placed at the respective Cα position (34). The energetics of the protein is described by a structure-based potential (35) (for details, see the Supporting text), which represents the funnel-like energy landscape for protein folding (35) and has been used extensively in studying protein-protein (36) and protein-nucleic acid interactions (37)(38)(39)(40)(41)(42)(43). The resolution of the ssDNA molecule is three beads per nucleotide (phosphate, sugar and nitrogenous base), placed at their respective geometric centers.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of Dynamic Binding of Replication Protein A to ssDNA

Bhattarcharjee

Mondal

2020

Preprint

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Replication protein A (RPA) serves as hub protein inside eukaryotic cells, where it coordinates crucial DNA metabolic processes and activates the DNA-damage response system. A characteristic feature of its action is to associate with ssDNA intermediates before handing over them to downstream proteins. The length of ssDNA intermediates differs for different pathways. This means RPA must have mechanisms for selective processing of ssDNA intermediates based on their length, the knowledge of which is fundamental to elucidate when and how DNA repair and replication processes are symphonized. By employing extensive molecular simulations, we investigated the mechanism of binding of RPA to ssDNA of different lengths. We show that the binding involves dynamic equilibrium with a stable intermediate, the population of which increases with the length of ssDNA. The vital underlying factors are decoded through collective variable principal component analysis. It suggests a differently orchestrated set of interactions that define the action of RPA based on the sizes of ssDNA intermediates. We further estimated the association kinetics and probed the diffusion mechanism of RPA to ssDNA. RPA diffuses on short ssDNA through progressive 'bulge' formation. With long ssDNA, we observed a conformational change in ssDNA coupled with its binding to RPA in a cooperative fashion. Our analysis explains how the 'short-lived,' long ssDNA intermediates are processed quickly in vivo. The study thus reveals the molecular basis of several recent experimental observations related to RPA binding to ssDNA and provides novel insights into the RPA functioning in DNA repair and replication. Significance StatementDespite ssDNA be the common intermediate to all pathways involving RPA, how does the latter function differently in the DNA processing events such as DNA repair, replication, and recombination just based on the length of ssDNA intermediates remains unknown. The major hindrance is the difficulty in capturing the transient interactions between the molecules. Even attempts to crystallize RPA complexes with 32nt and 62nt ssDNA have yielded a resolved structure of only 25nt ssDNA wrapped with RPA. Here, we used a state-of-the-art coarse-grained protein-ssDNA model to unravel the detailed mechanism of binding of RPA to ssDNA. Our study illustrates the molecular origin of variations in RPA action during various DNA processing events depending on the length of ssDNA intermediates.

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Reversible Intracellular Gelation of MCF10A Cells Enables Programmable Control Over 3D Spheroid Growth

McNally,

Macdougall,

Kirkpatrick

et al. 2023

Adv Healthcare Materials

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In nature, some organisms survive extreme environments by inducing a biostatic state wherein cellular contents are effectively vitrified. Recently, a synthetic biostatic state in mammalian cells is achieved via intracellular network formation using bio‐orthogonal strain‐promoted azide‐alkyne cycloaddition (SPAAC) reactions between functionalized poly(ethylene glycol) (PEG) macromers. In this work, the effects of intracellular network formation on a 3D epithelial MCF10A spheroid model are explored. Macromer‐transfected cells are encapsulated in Matrigel, and spheroid area is reduced by ≈50% compared to controls. The intracellular hydrogel network increases the quiescent cell population, as indicated by increased p21 expression. Additionally, bioenergetics (ATP/ADP ratio) and functional metabolic rates are reduced. To enable reversibility of the biostasis effect, a photosensitive nitrobenzyl‐containing macromer is incorporated into the PEG network, allowing for light‐induced degradation. Following light exposure, cell state, and proliferation return to control levels, while SPAAC‐treated spheroids without light exposure (i.e., containing intact intracellular networks) remain smaller and less proliferative through this same period. These results demonstrate that photodegradable intracellular hydrogels can induce a reversible slow‐growing state in 3D spheroid culture.

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Disparity in anomalous diffusion of proteins searching for their target DNA sites in a crowded medium is controlled by the size, shape and mobility of macromolecular crowders

Abstract: Using extensive computer simulations, we analyzed the role of physical properties of molecular crowding agents such as size, shape and mobility in the target search dynamics of DNA binding proteins.

Cited by 24 publications

References 59 publications

Structural Basis of Enhanced Facilitated Diffusion of DNA Binding Proteins in Crowded Cellular Milieu

Structural Basis of Enhanced Facilitated Diffusion of DNA Binding Proteins in Crowded Cellular Milieu

Mechanism of Dynamic Binding of Replication Protein A to ssDNA

Reversible Intracellular Gelation of MCF10A Cells Enables Programmable Control Over 3D Spheroid Growth

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