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Results for “Biophysics”

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bioRxiv2019

Nonreciprocal and Conditional Cooperativity Directs the Pioneer Activity of Pluripotency Transcription Factors

Cooperative binding of transcription factors (TFs) to chromatin orchestrates gene expression programming and cell fate specification. However the biophysical principles of TF cooperativity remain incompletely understood. Here we use single-molecule fluorescence microscopy to study the partnership between Sox2 and Oct4, two core members of the pluripotency gene regulatory network. We find that Sox2s pioneer activity (the ability to target DNA inside nucleosomes) is strongly affected by the translational and rotational positioning of its binding motif, while Oct4 can access nucleosomal sites with equal capacities. Furthermore, the Sox2-Oct4 pair displays nonreciprocal cooperativity, with Oct4 modulating Sox2s binding to the nucleosome but not vice versa. Such cooperativity is conditional upon the composite motif residing at specific nucleosomal locations. These results reveal that pioneer factors possess distinct properties of nucleosome targeting and suggest that the same set of TFs may differentially regulate transcriptional activity in a gene-specific manner on the basis of their motif positioning in the nucleosomal context.

biophysics↗

bioRxiv2019

Geometric state-space of protein-induced spontaneous curvature on helicoidal ramps in organelle membranes

Cellular membranes display an incredibly diverse range of shapes, both in the plasma membrane and at membrane bound organelles. These morphologies are intricately related to cellular functions, enabling and regulating fundamental membrane processes. However, the biophysical mechanisms at the origin of these complex geometries are not fully understood from the standpoint of membrane-protein coupling. In this work, we focused on a minimal model of helicoidal ramps representative of specialized endoplasmic reticulum compartments. Given a helicoidal membrane geometry, we asked what is the distribution of spontaneous curvature required to maintain this shape at mechanical equilibrium? Based on the Helfrich energy of elastic membranes with spontaneous curvature, we derived the shape equation for minimal surfaces, and applied it to helicoids. We showed the existence of switches in the sign of the spontaneous curvature associated with geometric variations of the membrane structures. Furthermore, for a prescribed gradient of spontaneous curvature along the exterior boundaries, we identified configurations of the helicoidal ramps that are confined between two infinitely large energy barriers. Overall our results suggest possible mechanisms for geometric control of helicoidal ramps in membrane organelles based on curvature-inducing proteins.

biophysics↗

bioRxiv2019

Characterization of Single Protein Dynamics in Cell Plasma Membrane Derived Polymer Cushioned Lipid Bilayers

Native cell membrane derived supported lipid bilayers (SLBs) are emerging platforms that have broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact of the bilayer membrane to the supporting substrate, and thus the mobility of transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins of distinct structures, i.e., GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescence proteins (GFPs) and their dynamics were measured by fluorescence single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length to protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1,000 to 5,000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were raised by increasing the polymer length, whereas the diffusion coefficient of GPI-anchored protein remained almost identical with different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 m2/s approximately) in the cushioned membrane with the longest polymer length (molecular weight of 5,000), indicating that the SLBs were fully suspended from the substrate by the polymer cushion at the microscopic length scale. Transient confinements were observed from all three proteins, and increasing the polymer length reduced the tendency of transient confinements. The measured dynamics of membrane proteins were found to be nearly unchanged after depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer cushioned plasma membrane bilayers that are potentially useful for future developments of membrane-based biosensors and analytical assays.

biophysics↗

bioRxiv2019

Identifying dynamic, partially occupied residues using anomalous scattering

X-ray crystallography is generally used to take single snapshots of a proteins conformation. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes Residual Anomalous and Electron Density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data collection and analysis strategy for partially occupied iodine anomalous signals. Using the long wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with as low as ~12% occupancy is demonstrated. The number and position of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly dynamic sections of crystal structures.\n\nSynopsisStructural studies on partially occupied, dynamic protein systems by crystallography are difficult. We present methods here for detecting these states in crystals.

biophysics↗

bioRxiv2019

Reconstructing wells from high density regions extracted from super-resolution single particle trajectories

Large amount of super-resolution single particle trajectories has revealed that the cellular environment is enriched in heterogenous regions of high density, which remain unexplained. The biophysical properties of these regions are characterized by a drift and their extension (a basin of attraction) that can be estimated from an ensemble of trajectories. We develop here two statistical methods to recover the dynamics and local potential wells (field of force and boundary) using as a model a truncated Ornstein-Ulhenbeck process. The first method uses the empirical distribution of points, which differs inside and outside the potential well, while the second focuses on recovering the drift field. Finally, we apply these two methods to voltage-gated calcium channels and phospholipids moving on the surface of neuronal cells and recover the energy and size of these high-density regions with nanometer precision.

biophysics↗

bioRxiv2019

Mechanosensitive junction remodelling promotes robust epithelial morphogenesis

Morphogenesis of epithelial tissues requires tight spatiotemporal coordination of cell shape changes. In vivo, many tissue-scale shape changes are driven by pulsatile contractions of intercellular junctions, which are rectified to produce irreversible deformations. The functional role of this pulsatory ratchet and its mechanistic basis remain unknown. Here we combine theory and biophysical experiments to show that mechanosensitive tension remodelling of epithelial cell junctions promotes robust epithelial shape changes via ratcheting. Using optogenetic control of actomyosin contractility, we find that epithelial junctions show elastic behaviour under low contractile stress, returning to their original lengths after contraction, but undergo irreversible deformation under higher magnitudes of contractile stress. Existing vertex-based models for the epithelium are unable to capture these results, with cell junctions displaying purely elastic or fluid-like behaviours, depending on the choice of model parameters. To describe the experimental results, we propose a modified vertex model with two essential ingredients for junction mechanics: thresholded tension remodelling and continuous strain relaxation. First, a critical strain threshold for tension remodelling triggers irreversible junction length changes for sufficiently strong contractions, making the system robust to small fluctuations in contractile activity. Second, continuous strain relaxation allows for mechanical memory removal, enabling frequency-dependent modulation of cell shape changes via mechanical ratcheting. Taken together, the combination of mechanosensitive tension remodelling and junctional strain relaxation provides a robust mechanism for large-scale morphogenesis.

biophysics↗

bioRxiv2019

The conical shape of DIM lipids promotes Mycobacterium tuberculosis infection of macrophages

Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen Mycobacterium tuberculosis (Mtb). While this lipid promotes the entry of Mtb into macrophages, which occurs via phagocytosis, its molecular mechanism of action is unknown. Here, we combined biophysical, cell biology, and modelling approaches to reveal the molecular mechanism of DIM action on macrophage membranes leading to the first step of Mtb infection. MALDI-TOF mass spectrometry showed that DIM molecules are transferred from the Mtb envelope to macrophage membranes during infection. Multi-scale molecular modeling and 31P-NMR experiments revealed that DIM adopts a conical shape in membranes and aggregate in the stalks formed between two opposing lipid bilayers. Infection of macrophages pre-treated with lipids of various shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken together, these results reveal how the molecular shape of a mycobacterial lipid can modulate the biological function of macrophages.

biophysics↗

bioRxiv2019

Nanomechanics and co-transcriptional folding of Spinach and Mango

Recent advances in fluorogen-binding RNA aptamers known as \"light-up\" aptamers provide an avenue for protein-free detection of RNA in cells. Crystallographic studies have revealed a G-Quadruplex (GQ) structure at the core of light-up aptamers such as Spinach, Mango and Corn. Detailed biophysical characterization of folding of such aptamers is still lacking despite the potential implications on their in vivo folding and function. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolded in four discrete steps as force is increased to 7 pN and refolded in reciprocal steps upon force relaxation. Binding of DFHBI-1T fluorogen preserved the step-wise unfolding behavior although at slightly higher forces. In contrast, GQ core unfolding in iMangoIII and MangoIV occurred in one discrete step at forces > 10 pN and refolding occurred at lower forces showing hysteresis. Binding of the cognate fluorogen, TO1, did not significantly alter the mechanical stability of Mangos. In addition to K+, which is needed to stabilize the GQ cores, Mg2+ was needed to obtain full mechanical stability of the aptamers. Co-transcriptional folding analysis using superhelicases showed that co-transcriptional folding reduces misfolding and allows a folding pathway different from refolding. As the fundamental cellular processes like replication, transcription etc. exert pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold.

biophysics↗

bioRxiv2019

Error-speed correlations in biopolymer synthesis

Synthesis of biopolymers such as DNA, RNA, and proteins are biophysical processes aided by enzymes. Performance of these enzymes is usually characterized in terms of their average error rate and speed. However, because of thermal fluctuations in these single-molecule processes, both error and speed are inherently stochastic quantities. In this paper, we study fluctuations of error and speed in biopolymer synthesis and show that they are in general correlated. This means that, under equal conditions, polymers that are synthesized faster due to a fluctuation tend to have either better or worse errors than the average. The error-correction mechanism implemented by the enzyme determines which of the two cases holds. For example, discrimination in the forward reaction rates tends to grant smaller errors to polymers with faster synthesis. The opposite occurs for discrimination in monomer rejection rates. Our results provide an experimentally feasible way to identify error-correction mechanisms by measuring the error-speed correlations.

biophysics↗

bioRxiv2019

Streamlining effects of extra telomeric repeat on telomere folding revealed by fluorescence-force spectroscopy

Tandem repeats of guanine rich sequences are ubiquitous in the eukaryotic genome. For example, in the human cells, telomeres at the chromosomal ends comprise of kilobases repeats of T2AG3. Four such repeats can form G-quadruplexes (GQs). Biophysical studies have shown that GQs formed from four consecutive repeats possess high diversity both in their structure and in their response to tension. In principle, a GQ can form from any four repeats that may not even be consecutive. In order to investigate the dynamics of GQ possessing such positional multiplicity, we studied five and six repeats human telomeric sequence using single molecule FRET as well as its combination with optical tweezers. Our results suggest preferential formation of GQs at the 3 end both in K+ and Na+ solutions although minority populations with a 5 GQ or long-loop GQs were also observed. Using a vectorial folding assay which mimics the directional nature of telomere extension, we found that the 3 preference holds even when folding is allowed to begin from the 5 side. Interestingly, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. Location of GQ on a long G-rich telomeric overhang and reduction in diversity of GQ conformations and mechanical responses through adjacent sequences have important implications in processes such as telomerase inhibition, alternative lengthening of telomeres, T-loop formation, telomere end protection and replication.

biophysics↗

bioRxiv2019

NMR-based analysis of nucleotide π-stacking in a crowded environment: Implications for prebiotic reactions

The inherent heterogeneity of the prebiotic milieu is often overlooked when studying nonenzymatic reactions. However, it is important to note that the prebiotic soup of a putative RNA World would have been replete with a plethora of molecules resulting from complex chemical syntheses, as well as exogeneous delivery. The presence of such background molecules could lead to pertinent phenomenon such as molecular crowding, which can potentially affect how a reaction would advent in a crowded milieu. In the current study, we have analyzed the effect of crowding on the stacking ability of the RNA monomers, using Nuclear Magnetic Resonance (NMR) spectroscopy. Our findings corroborate that the purine monomers possess better stacking efficiency than pyrimidine based monomers. Significantly, this competence is further enhanced in the presence of a crowding agent. Interestingly, this enhanced stacking could result in higher sequestration of the purine monomers, putting their ready availability for relevant nonenzymatic polymerization and replication reactions into question. Taken together, this study demonstrates the need for systematic biophysical characterization of molecular crowding in the context of prebiotically pertinent processes. Unravelling such phenomena is essential to gather a real understanding of how the transition from abiotic to biotic, would have happened during the origin of life.

biophysics↗

bioRxiv2019

Elucidating the Influence of Linker Histone Isoforms on Chromatosome Dynamics and Energetics

Linker histones are epigenetic regulators that bind to nucleosomes and alter chromatin structures and dynamics. Biophysical studies have revealed two binding modes in the linker histone/nucleosome complex, the chromatosome, where the linker histone is either centered on or askew from the dyad axis. Each has been posited to have distinct effects on chromatin, however the molecular and thermodynamic mechanisms that drive them and their dependence on linker histone compositions remain poorly understood. We present molecular dynamics simulations of chromatosomes with the globular domain of two linker histone variants, generic H1 (genGH1) and H1.0 (GH1.0), to determine how their differences influence chromatosome structures, energetics, and dynamics. Results show that both unbound linker histones adopt a single compact conformation. Upon binding, DNA flexibility is reduced, resulting in increased chromatosome compaction. While both variants enthalpically favor on-dyad binding, energetic benefits are significantly higher for GH1.0, suggesting that GH1.0 is more capable than genGH1 of overcoming the large entropic reduction required for on-dyad binding which helps rationalize experiments that have consistently demonstrated GH1.0 in on-dyad states but that show genGH1 in both locations. These simulations highlight the thermodynamic basis for different linker histone binding motifs, and details their physical and chemical effects on chromatosomes.

biophysics↗

bioRxiv2019

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix, which includes the Golgi Reassembly and Stacking Proteins (GRASPs), which participate in cisternae stacking and lateral linkage in vertebrates. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different protein binding partners. The conserved N-terminus of the GRASP family includes two PDZ domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content, however PDZ1 alone mediates nearly all the interactions between GRASPs and their binding partners. In this work, NMR, Synchrotron-Radiation Circular Dichroism and Molecular Dynamics were used to examine the structure, flexibility and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual {beta}31{beta}4{beta}52{beta}6{beta}1{beta}2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable {beta}2-strand and a more flexible and unstable 2-helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using Molecular Dynamics simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.\n\nSignificance StatementGolgi Reassembly and Stacking Proteins (GRASPs) play pivotal roles in the maintenance of Golgi structure as well as in unconventional protein secretion. Their broad network of interactions is mainly sustained by the two-PDZ domains located in the N-terminal portion of the protein. The asymmetry of the PDZ domains in terms of number and diversity of interacting partners has been long recognized, but the molecular determinants of that asymmetry remains largely unknown. The biophysical data presented here provide a firm basis for understanding why PDZ1 behaves differently to PDZ2 in solution, despite their similar 3D structures. Furthermore, we propose that PDZ2 assist ligand binding to PDZ1, by means of conformational stabilization.

biophysics↗

bioRxiv2019

Analyzing and comparing the radius of gyration and hydrodynamic radius in conformational ensembles of intrinsically disordered proteins

The level of compaction of an intrinsically disordered protein may affect both its physical and biological properties, and can be probed via different types of biophysical experiments. Small-angle X-ray scattering (SAXS) probe the radius of gyration (Rg) whereas pulsed-field-gradient nuclear magnetic resonance (NMR) diffusion, fluorescence correlation spectroscopy and dynamic light scattering experiments can be used to determine the hydrodynamic radius (Rh). Here we show how to calculate Rg and Rh from a computationally-generated conformational ensemble of an intrinsically disordered protein. We further describe how to use a Bayesian/Maximum Entropy procedure to integrate data from SAXS and NMR diffusion experiments, so as to derive conformational ensembles in agreement with those experiments.

biophysics↗

bioRxiv2019

PEGylated surfaces for the study of DNA-protein interactions by atomic force microscopy

DNA-protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA-protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working at biologically relevant protein concentrations, where protein may be present at large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113 / PLL1000-2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show that this selective adsorption extends to DNA-protein complexes, through the use of biotinylated DNA and streptavidin, and demonstrate that DNA-bound streptavidin can be unambiguously distinguished by in-liquid AFM in spite of an excess of unbound streptavidin in solution.

biophysics↗

bioRxiv2019

Reorganisation of complex ciliary flows around regenerating Stentor coeruleus

The phenomenon of ciliary coordination has garnered increasing attention in recent decades, with multiple theories accounting for its emergence in different contexts. The heterotrich ciliate Stentor coeruleus is a unicellular organism which boasts a number of features which present unrivalled opportunities for biophysical studies of cilia coordination. With their cerulean colour and distinctive morphology, these large protists possess a characteristic differentiation between cortical rows of short body cilia used for swimming, and an anterior ring structure of fused oral cilia forming a membranellar band. The oral cilia beat metachronously to produce strong feeding currents. In addition to this complex body plan, Stentor have remarkable regenerative capabilities. Minute fragments of single cells can over the period of hours or days, regenerate independently into new, proportional individuals. Certain environmental perturbations elicit a unique programmed response known as oral regeneration wherein only the membranellar band is shed and a new, ciliated oral primordium formed on the side of the body. Here, we target oral regeneration induced by sucrose-shock to reveal the complex interplay between ciliary restructuring and hydrodynamics in Stentor, which accompanies the complete developmental sequence from band formation, elongation, curling, and migration toward the cell anterior.\n\n\"When the anterior part is open, one may perceive about its Edges a very lively Motion; and when the Polyps presents itself in a certain manner, it discovers, on either side of these edges of its anterior part, somewhat very much resembling the wheels of a little Mill, that move with great velocity.\"\n\nA. Trembley F.R.S describing the membranellar band of Stentor, Phil. Soc. Trans. Royal Society (London), 1744.

biophysics↗

bioRxiv2019

Human POT1 Unfolds G-Quadruplexes by Conformational Selection

The reaction mechanism by which shelterin protein POT1 (Protection of Telomeres) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. We show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is fast, with an apparent relaxation time of 80.0 {+/-} 0.4 ms, and strong, with a binding free energy of -10.1 {+/-} 0.3 kcal mol-1. That favourable free energy arises from a large favourable enthalpy contribution of -38.2 {+/-} 0.3 kcal mol-1. In sharp contrast, the binding of POT1 to an initially folded 24 nt G-quadruplex structure is slow, with an average relaxation time of 2000-3000 s. Fluorescence, circular dichroism and analytical ultracentrifugation studies show that POT1 binding is coupled to quadruplex unfolding with a final stoichiometry of 2 POT1 molecules bound per 24 nt DNA. The binding isotherm for the POT1-quadruplex binding interaction is sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provides an excellent quantitative fit to the experimental binding data. The overall favourable free energy of the POT1-quadruplex interaction is -7.1 kcal mol-1, which arises from a balance between unfavourable free energy of +3.4 kcal mol-1 for quadruplex unfolding and a large, favorable free energy of -10.5 kcal mol-1 for POT1 binding. We show that POT1 can unfold and bind to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid or parallel), but will not interact with duplex DNA or with a parallel G-quadruplex structure formed by a c-myc promoter sequence. Finally, molecular dynamics simulations provide a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

biophysics↗

bioRxiv2019

Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen

Forster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra- and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We then tested the red FPs ability to act as FRET acceptors using mNeonGreen-Red FP tandem construct. With these constructs we found that mScarlet-I and mCherry are able to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these same assays. We explore possible explanations for this poor performance but are unable to definitively determine the cause, all though protein maturation seems to play a role. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with mScarlet-I out performing mCherry due its higher brightness.

biophysics↗