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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 54,615 bioRxiv papers from 252,242 authors.

Most downloaded bioRxiv papers, since beginning of last month

in category biophysics

2,200 results found. For more information, click each entry to expand.

61: The deadly touch: protein denaturation at the water-air interface and how to prevent it
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Posted to bioRxiv 25 Aug 2018

The deadly touch: protein denaturation at the water-air interface and how to prevent it
106 downloads biophysics

Edoardo D'Imprima, Davide Floris, Mirko Joppe, Ricardo Sánchez, Martin Grininger, Werner Kühlbrandt

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation of the protein. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions are those facing the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

62: Spontaneous Self-assembly of Amyloid β (1-40) into Dimers
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Posted to bioRxiv 04 Jun 2019

Spontaneous Self-assembly of Amyloid β (1-40) into Dimers
104 downloads biophysics

Mohtadin Hashemi, Yuliang Zhang, Zhengjian Lv, Yuri L Lyubchenko

The self-assembly and fibrillation of amyloid β (Aβ) proteins is the neuropathological hallmark of Alzheimer's disease. However, the molecular mechanism of how disordered monomers assemble into aggregates remains largely unknown. In this work, we characterize the assembly of Aβ(1-40) monomers into dimers using long-time molecular dynamics simulations. Upon interaction, the monomers undergo conformational transitions, accompanied by change of the structure, leading to the formation of a stable dimer. The dimers are primarily stabilized by interactions in the N-terminal region (residues 5-12), in the central hydrophobic region (residues 16-23), and in the C-terminal region (residues 30-40); with inter-peptide interactions focused around the N- and C- termini. The dimers do not contain long β-strands that are usually found in fibrils.

63: Magnetic-alignment of polymer macro-nanodiscs enable residual dipolar couplings based high-resolution structural studies by NMR
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Posted to bioRxiv 14 Jun 2019

Magnetic-alignment of polymer macro-nanodiscs enable residual dipolar couplings based high-resolution structural studies by NMR
103 downloads biophysics

Thirupathi Ravula, Ayyalusamy Ramamoorthy

Residual dipolar couplings (RDCs) have been shown to be valuable for the structural studies of systems ranging from small molecules to large proteins. Here we demonstrate the lyotropic liquid crystal behavior of polymer macro-nanodiscs (> 20 nm in diameter) and enable the measurement of RDCs using high resolution NMR.

64: Structure of a cytochrome-based bacterial nanowire
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Posted to bioRxiv 10 Dec 2018

Structure of a cytochrome-based bacterial nanowire
103 downloads biophysics

David J Filman, Stephen F Marino, Joy E Ward, Lu Yang, Zoltan Mester, Esther Bullitt, Derek R Lovley, Mike Strauss

Electrically conductive pili from Geobacter species, termed bacterial - nanowires -, are intensely studied for their biological significance and potential in the development of new materials. We have characterized a unique nanowire from conductive G. sulfurreducens pili preparations by cryo-electron microscopy composed solely of the c-type cytochrome OmcS. We present here, at 0.34 nm resolution, a novel structure of a cytochrome-based filament and discuss its possible role in long-range biological electron transport.

65: Energy landscapes reveal agonist's control of GPCR activation via microswitches
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Posted to bioRxiv 04 May 2019

Energy landscapes reveal agonist's control of GPCR activation via microswitches
102 downloads biophysics

Oliver Fleetwood, Pierre Matricon, Jens Carlsson, Lucie Delemotte

Agonist binding to the extracellular part of G protein-coupled receptors (GPCRs) leads to conformational changes in the transmembrane region that activate cytosolic signalling pathways. Although high resolution structures of the inactive and active receptor states are available, the allosteric coupling that transmits the signal across the membrane is not fully understood. We calculated free energy landscapes of the β2 adrenergic receptor using atomistic molecular dynamics simulations in an optimized string-of-swarms framework, which sheds new light on the roles of microswitches involved in activation. Contraction of the extracellular binding site in the presence of agonist is obligatorily coupled to conformational changes in a connector motif located in the core of the transmembrane region. In turn, the connector is probabilistically coupled to the conformation of the intracellular region. An active connector promotes desolvation of a buried solvent-filled cavity and a twist of the conserved NPxxY motif, which leads to a larger population of active-like states at the G protein binding site. This effect is further augmented by protonation of the strongly conserved Asp79, which locks the NPxxY motif and solvent cavity in active-like conformations. The agonist binding site hence communicates with the intracellular region via a cascade of locally connected switches and the free energy landscapes along these contributes to understanding of how ligands can stabilize distinct receptor states. We demonstrate that the developed simulation protocol is transferable to other class A GPCRs and anticipate that it will become a useful tool in design of drugs with specific signaling properties.

66: High-yield fabrication of DNA and RNA scaffolds for single molecule force and torque spectroscopy experiments.
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Posted to bioRxiv 06 Jun 2019

High-yield fabrication of DNA and RNA scaffolds for single molecule force and torque spectroscopy experiments.
102 downloads biophysics

Flavia Stal Papini, Mona Seifert, David Dulin

Single molecule biophysics experiments have enabled the observation of biomolecules with a great deal of precision in space and time, e.g. nucleic acids mechanical properties and protein-nucleic acids interactions using force and torque spectroscopy techniques. The success of these experiments strongly depends on the capacity of the researcher to design and fabricate complex nucleic acid scaffolds, as the pertinence and the yield of the experiment strongly depend on the high quality and purity of the final scaffold. Though the molecular biology techniques involved are well known, the fabrication of nucleic acids scaffold for single molecule experiments still remains a difficult task. Here, we present new protocols to generate high quality coilable double-stranded DNA and RNA, as well as DNA and RNA hairpins with ~500-1000 bp long stems. Importantly, we present a new approach based on single-stranded DNA's annealing and show, using magnetic tweezers, that it is more efficient to generate complex nucleic acid scaffolds in larger amount and at higher purity than a standard PCR-digestion-ligation approach. The protocols we describe here enable the design of any sort of complex nucleic acid scaffold for single molecule biophysics experiments and will therefore be extremely valuable to the community.

67: Past-future information bottleneck framework for simultaneously sampling biomolecular reaction coordinate, thermodynamics and kinetics
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Posted to bioRxiv 28 Dec 2018

Past-future information bottleneck framework for simultaneously sampling biomolecular reaction coordinate, thermodynamics and kinetics
102 downloads biophysics

Yihang Wang, Joao Ribeiro, Pratyush Tiwary

The ability to rapidly learn from high-dimensional data to make reliable bets about the future outcomes is crucial in many contexts. This could be a fly avoiding predators, or the retina processing gigabytes of data almost instantaneously to guide complex human actions. In this work we draw parallels between such tasks, and the efficient sampling of complex biomolecules with hundreds of thousands of atoms. For this we use the Predictive Information Bottleneck (PIB) framework developed and used for the first two classes of problems, and re-formulate it for the sampling of biomolecular structure and dynamics, especially when plagued with rare events. Our method considers a given biomolecular trajectory expressed in terms of order parameters or basis functions, and uses a deep neural network to learn the minimally complex yet most predictive aspects of this trajectory, viz the PIB. This information is used to perform iterative rounds of biased simulations that enhance the sampling along the PIB to gradually improve its accuracy, directly obtaining associated thermodynamic and kinetic information. We demonstrate the method on two test-pieces, including benzene dissociation from the protein lysozyme, where we calculate the dissociation pathway and timescales slower than milliseconds. Finally, by performing an analysis of residues contributing to the PIB, we predict the critical mutations in the system which would be most impactful on the stability of the crucial but ephemeral transition state. We believe this work marks a big step forward in the use of predictive artificial intelligence ideas for the sampling of biomolecules.

68: Non-uniform distribution of myosin-mediated forces governs red blood cell curvature through tension modulation
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Posted to bioRxiv 12 Jun 2019

Non-uniform distribution of myosin-mediated forces governs red blood cell curvature through tension modulation
102 downloads biophysics

Haleh Alimohamadi, Alyson S Smith, Velia M Fowler, Padmini Rangamani

The biconcave disk shape of the mammalian red blood cell (RBC) is unique to the RBC and is vital for its circulatory function. Recent experiments have demonstrated that the biconcave shape of the RBC relies not only on the physical properties of the membrane but also depends on the molecular constituents of the membrane cytoskeleton, including the contractile activity of the nonmuscle myosin IIA (NMIIA) motor protein. Here, we use the classical Helfrich model for the RBC membrane and incorporate heterogeneous force distributions along the membrane to mimic the contractile activity of NMIIA. We find that the biconcave shape of the RBC depends on the ratio of forces per unit volume in the dimple and donut regions of the RBC. Experimental measurements of NMIIA densities at the dimple and donut validate our prediction that (a) membrane forces must be non-uniform along the RBC membrane and (b) the force density must be larger in the dimple region than the donut region to produce the observed membrane curvatures. Furthermore, we find that the tension of the RBC membrane plays an important role in regulating this force-shape landscape. Our findings of heterogeneous force distributions on the plasma membrane for RBC shape maintenance have implications for shape maintenance of many cell types.

69: Mathematical Methods for Modeling Chemical Reaction Networks
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Posted to bioRxiv 18 Aug 2016

Mathematical Methods for Modeling Chemical Reaction Networks
101 downloads biophysics

Justin Carden, Casian Pantea, Gheorge Craciun, Raghu Machiraju, Parag Mallick

Cancer's cellular behavior is driven by alterations in the processes that cells use to sense and respond to diverse stimuli. Underlying these processes are a series of chemical processes (enzyme-substrate, protein-protein, etc.). Here we introduce a set of mathematical techniques for describing and characterizing these processes.

70: Robin Hood: non-fitting, non-smoothing image detrending for bleaching correction
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Posted to bioRxiv 14 Jun 2019

Robin Hood: non-fitting, non-smoothing image detrending for bleaching correction
99 downloads biophysics

Rory Nolan, Maro Iliopoulou, Christian Siebold, E Yvonne Jones, Sergi Padilla-Parra

Recent advances in protein labelling-gene tagging with CRIPSR-Cas9-have made it possible to label proteins of interest endogenously. This represents a major breakthrough in the field of quantitative microscopy, especially when quantifying protein-protein interactions. This is because over-expression of labelled proteins may cause a distortion in localization, function and perhaps artificially force protein-protein interactions due to crowding effects. A microscopy technique that is particularly well suited to detect protein interactions with low photon budgets is number and brightness (N&B). Detrending (removal of global trends in data) is a necessary pre-processing step to N&B calculations, but all current detrending methods perform poorly at low intensities. Here, we present the Robin Hood automatic detrending algorithm which performs well at low intensities, evaluating it with simulated and low photon budget live cell images. RH is available as an ImageJ plugin and as an R package.

71: Intramolecular Communication and Allosteric Sites in Enzymes Unraveled by Time-Dependent Linear Response Theory
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Posted to bioRxiv 21 Jun 2019

Intramolecular Communication and Allosteric Sites in Enzymes Unraveled by Time-Dependent Linear Response Theory
98 downloads biophysics

Bang-Chieh Huang, Chi-Hong Chang-Chein, Lee-Wei Yang

It has been an established idea in recent years that protein is a physiochemically connected network. Allostery, understood in this new context, is a manifestation of residue communicating between remote sites in this network, and hence a rising interest to identify functionally relevant communication pathways and the frequent communicators within. Previous studies rationalized the coupling between functional sites and experimentally observed allosteric sites by theoretically discovered high positional/velocity/thermal correlations between these sites. However, for one to systematically discover previously unobserved allosteric sites in any receptor/enzyme providing the position of functional (orthosteric) sites, these high correlations may not be able to identify remote allosteric sites because of a number of false-positives while many of those are located in proximity to the functional site. Also, whether allosteric sites should be found in equilibrium or non-equilibrium state of a protein to be more biologically relevant is not clear, neither is the directionality preference of aforementioned propagating signals. In this study, we devised a time-dependent linear response theory (td-LRT) integrating intrinsic protein dynamics and perturbation forces that excite protein’s temporary reconfiguration at the non-equilibrium state, to describe atom-specific time responses as the propagating mechanical signals and discover that the most frequent remote communicators can be important allosteric sites, mutation of which would deteriorate the hydride transfer rate in DHFR by 2 to 3 orders. The preferred directionality of the signal propagation can be inferred from the asymmetric connection matrix (CM), where the coupling strength between a pair of residues is suggested by their communication score (CS) in the CM, which is found consistent with experimentally characterized nonadditivity of double mutants. Also, the intramolecular communication centers (ICCs), having high CSs, are found evolutionarily conserved, suggesting their biological importance.

72: Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen
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Posted to bioRxiv 05 Jul 2019

Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen
97 downloads biophysics

Tyler William McCullock, David M. MacLean, Paul Kammermeier

Förster 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 FP’s 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 it’s higher brightness.

73: Elucidating the Influence of Linker Histone Isoforms on Chromatosome Dynamics and Energetics
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Posted to bioRxiv 04 Jun 2019

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

Dustin C Woods, Jeff Weresczczynski

Linker histones are epigentic 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 two linker histone isoforms, globular H1 (GH1) and H5 (GH5), to determine how their differences influence chromatosome structures, energetics, and dynamics. Results show that both linker histones adopt a single compact conformation in solution. Upon binding, DNA flexibility is reduced and there is increased chromatosome compaction. While both isoforms favor on-dyad binding, the enthalpic benefit is significantly higher for GH5. This suggests that GH5 is more capable of overcoming the large entropic reduction required for on-dyad binding than GH1, which helps rationalize experiments that have consistently demonstrated GH5 in on-dyad states but that show GH1 in both locations. These simulations highlights the thermodynamic basis for different linker histone binding motifs, and details their physical and chemical effects on chromatosomes.

74: Molecular Dynamics Simulations, Exact NOE Measurements, and Machine Learning Reveal a Low-populated State of the UUCG RNA Tetraloop
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Posted to bioRxiv 02 Jul 2019

Molecular Dynamics Simulations, Exact NOE Measurements, and Machine Learning Reveal a Low-populated State of the UUCG RNA Tetraloop
97 downloads biophysics

Sandro Bottaro, Parker J. Nichols, Beat Vögeli, Michele Parrinello, Kresten Lindorff-Larsen

Many RNA molecules are dynamic, but characterizing their motions by experiments is often diZcult, often requiring application of complex NMR experiments. Computational methods such as molecular dynamics simulations, on the other hand, still suffer from diZculties in sampling and remaining force 1eld errors. Here, we provide an atomic-detailed description of structure and dynamics of the 14-mer UUCG RNA stem-loop by combining molecular dynamics simulations with exact nuclear Overhauser enhancement data. The integration of experiments and simulation via a Bayesian/Maximum entropy approach enable us to discover and characterize a new state of this molecule, which we show samples two distinct states. The most stable conformation corresponds to the native, consensus three-dimensional structure. The second, low-populated state is characterized by the absence of the peculiar non-Watson-Crick base pair between U and G in the loop region. We use machine learning methods to analyse the MD simulation, that reveal the presence of speci1c proton-proton contacts that characterize the alternative loop conformation. We identify these contacts in the NOESY spectrum, thereby providing evidence for the existence of the low-populated state. Together, our results demonstrate a new approach to determine the structure and thermodynamics of conformational changes in RNA molecules.

75: Wavelength-encoded laser particles for massively-multiplexed cell tagging
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Posted to bioRxiv 07 Nov 2018

Wavelength-encoded laser particles for massively-multiplexed cell tagging
97 downloads biophysics

Nicola Martino, Sheldon J.J. Kwok, Andreas C. Liapis, Sarah Forward, Hoon Jang, Hwi-Min Kim, Sarah J. Wu, Jiamin Wu, Paul H. Dannenberg, Sun-Joo Jang, Yong-Hee Lee, Seok-Hyun Yun

Large-scale single-cell analyses have become increasingly important given the role of cellular heterogeneity in complex biological systems. However, no current techniques enable optical imaging of uniquely-tagged individual cells. Fluorescence-based approaches can only distinguish a handful of distinct cells or cell groups at a time because of spectral crosstalk between conventional fluorophores. Here we show a novel class of imaging probes emitting coherent laser light, called laser particles. Made of silica-coated semiconductor microcavities, these laser particles have single-mode emission over a broad range from 1170 to 1580 nm with sub-nm linewidths, enabling massive spectral multiplexing. We demonstrate the stability and biocompatibility of these probes in vitro and their utility for wavelength-multiplexed cell tagging and imaging. We demonstrate real-time tracking of thousands of individual cells in a 3D tumor model for several days showing different behavioral phenotypes. We expect laser particles will enable new approaches for single-cell analyses.

76: Myosin-II mediated traction forces evoke localized Piezo1 Ca2+ flickers
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Posted to bioRxiv 03 Apr 2018

Myosin-II mediated traction forces evoke localized Piezo1 Ca2+ flickers
96 downloads biophysics

Kyle Ellefsen, Jesse R Holt, Alice Chang, Jamison L. Nourse, Janahan Arulmoli, Armen Mekhdjian, Hamid Abuwarda, Francesco Tombola, Lisa A Flanagan, Alexander R Dunn, Ian Parker, Medha M Pathak

Piezo channels transduce mechanical stimuli into electrical and chemical signals, and in doing so, powerfully influence development, tissue homeostasis, and regeneration. While much is known about how Piezo1 responds to external forces, its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that actomyosin-based cellular traction forces generate spatially-restricted Ca2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched in regions proximal to force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that diffusion allows channel molecules to efficiently respond to transient, local mechanical stimuli.

77: Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase
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Posted to bioRxiv 26 Sep 2018

Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase
96 downloads biophysics

Alexandrea N Rizo, JiaBei Lin, Stephanie N. Gates, Eric Tse, Stephen M Bart, Laura M Castellano, Frank DiMaio, James Shorter, Daniel R Southworth

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. To elucidate the translocation mechanism, we determined the cryo-EM structure of a hyperactive ClpB variant to 2.9 angstrom resolution bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS. Distinct substrate-gripping mechanisms are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent the topmost NBD1 contact. NBD conformations at the spiral seam reveal how ATP hydrolysis and substrate engagement or disengagement are precisely tuned to drive a stepwise translocation cycle.

78: Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets
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Posted to bioRxiv 18 Nov 2018

Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets
95 downloads biophysics

Serena Sanulli, Mike J Trnka, Venkat Dharmarajan, Ryan W Tibble, Bruce D Pascal, Alma L Burlingame, Patrick R Griffin, John D Gross, Geeta J. Narlikar

The heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet formation. Yet, a connection between phase separation and chromatin compaction has not been experimentally demonstrated. More fundamentally, how HP1 action at the level of a single nucleosome drives chromatin compaction remains poorly understood. Here we directly demonstrate that the S. pombe HP1 protein, Swi6, compacts arrays of multiple nucleosomes into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a mononucleosome. Restraining these dynamics via site-specific disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. Our results indicate that chromatin compaction and phase separation can be highly coupled processes. Further, we find that such coupling is promoted by a counter-intuitive function of Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by weak and dynamic multivalent interactions. We propose that dynamic exposure of buried histone residues increases opportunities for multivalent interactions between nucleosomes, thereby coupling chromatin compaction to phase separation. We anticipate that this new model for chromatin organization may more generally explain the formation of highly compacted chromatin assemblies beyond heterochromatin.

79: Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome
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Posted to bioRxiv 10 Jun 2019

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

Luis F. S. Mendes, Mariana R. B. Batista, Peter J Judge, Anthony Watts, Christina Redfield, Antonio J Costa-Filho

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 β3α1β4β5α2β6β1β2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable β2-strand and a more flexible and unstable α2-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.

80: Liquid-Liquid Phase Separation is Driven by Large-Scale Conformational Unwinding and Fluctuations of Intrinsically Disordered Protein Molecules
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Posted to bioRxiv 29 Apr 2019

Liquid-Liquid Phase Separation is Driven by Large-Scale Conformational Unwinding and Fluctuations of Intrinsically Disordered Protein Molecules
92 downloads biophysics

Anupa Majumdar, Priyanka Dogra, Shiny Maity, Samrat Mukhopadhyay

Liquid-liquid phase separation occurs via a multitude of transient, non-covalent, intermolecular interactions resulting in phase transition of intrinsically disordered proteins/regions (IDPs/IDRs) and other biopolymers into mesoscopic, dynamic, non-stoichiometric, supramolecular condensates. IDPs resemble associative polymers possessing stereospecific "stickers" and flexible "spacers" that govern the transient chain-chain interactions and fluidity in phase-separated liquid droplets. However, the fundamental molecular origin of phase separation remains elusive. Here we present a unique case to demonstrate that unusual conformational expansion events coupled with solvation and fluctuations drive phase separation of tau, an IDP associated with Alzheimer's disease. Using intramolecular excimer emission as a powerful proximity readout, we show the unraveling of polypeptide chains within the protein-rich interior environment that can promote critical interchain contacts. Using highly-sensitive picosecond time-resolved fluorescence depolarization measurements, we directly capture rapid large-amplitude torsional fluctuations in the extended chains that can control the relay of making-and-breaking of noncovalent intermolecular contacts maintaining the internal fluidity. Our observations, together with the existing polymer theories, suggest that such an orchestra of concerted molecular shapeshifting events involving chain expansion, solvation, and fluctuations can provide additional favorable free energies to overcome the entropy of mixing term during phase separation. The interplay of these key molecular parameters can also be of prime importance in modulating the mesoscale material property of liquid-like condensates and their maturation of into pathological gel-like and solid-like aggregates.

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