Featured
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Research Briefing |
Identifying phase-separating biomolecular condensates in cells
We developed a high-throughput, unbiased strategy for the identification of endogenous biomolecular condensates by merging cell volume compression, sucrose density gradient centrifugation and quantitative mass spectrometry. We demonstrated the performance of this strategy by identifying both global condensate proteins and those responding to specific biological processes on a proteome-wide scale.
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Article |
High-throughput and proteome-wide discovery of endogenous biomolecular condensates
High-throughput proteome-wide methods for identifying endogenous proteins that phase separate or partition into condensates during certain physiological events are needed but remain a challenge. Now, a high-throughput, unbiased and quantitative strategy can identify endogenous biomolecular condensates and screen proteins involved in phase separation on a proteome-wide scale.
- Pengjie Li
- , Peng Chen
- & Yiwei Li
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Article
| Open AccessRNA modulates hnRNPA1A amyloid formation mediated by biomolecular condensates
The underlying mechanism for how heterotypic protein–RNA interactions modulate the liquid to amyloid transition of hnRNPA1A, a protein involved in amyotrophic lateral sclerosis, has so far remained elusive. Now characterization of hnRNPA1A condensate formation and aggregation in vitro reveals that the RNA/protein stoichiometry affects the molecular pathways leading to amyloid formation.
- Chiara Morelli
- , Lenka Faltova
- & Paolo Arosio
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Article
| Open AccessAsymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility
The biomolecular principles underlying the formation of multiphasic condensates have been difficult to elucidate owing to a paucity of tools, especially within living cells. In this work synthetic orthogonal protein scaffolds alongside molecular simulations are used to highlight how the oligomerization of disordered proteins can asymmetrically drive miscibility–immiscibility transitions.
- Ushnish Rana
- , Ke Xu
- & Clifford P. Brangwynne
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Article
| Open AccessCharacterizing ATP processing by the AAA+ protein p97 at the atomic level
The human ATP-hydrolysing enzyme p97 populates a metastable reaction intermediate, the ADP·Pi state, which is poised between hydrolysis and product release. Now, molecular motions at the active site in the temporal window immediately before and after ATP hydrolysis have been elucidated by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations.
- Mikhail Shein
- , Manuel Hitzenberger
- & Anne K. Schütz
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Article |
Determinants that enable disordered protein assembly into discrete condensed phases
Cells spatially organize biochemical reactions within membrane-bound and membraneless compartments. The extent to which intrinsically disordered proteins themselves can form discrete compartments or condensed phases is poorly understood. Now a pair of model IDRs that display orthogonality in condensation and the chain features governing selective assembly have been identified.
- Rachel M. Welles
- , Kandarp A. Sojitra
- & Matthew C. Good
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News & Views |
How membranes influence intracellular phase separation
Liquid–liquid phase separation (LLPS) within cells is a captivating phenomenon known to aid the organization of cellular components; however, its complex kinetics have remained a puzzle. Now, a new study elucidates the crosstalk between the phase state of an encapsulating membrane and LLPS dynamics.
- Rumiana Dimova
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Article |
Subdomain dynamics enable chemical chain reactions in non-ribosomal peptide synthetases
Many natural products are produced by non-ribosomal peptide synthetases in an assembly-line fashion. How these molecular machines orchestrate the biochemical sequences has remained elusive. It is now understood that an extended-conformation ensemble is needed to coordinate chemical-transformation steps whereas the biosynthesis directionality is driven by the enzyme’s innate conformational free energies.
- Xun Sun
- , Jonas Alfermann
- & Haw Yang
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News & Views |
Using symmetry to drive new protein assemblies
Recent improvements in de novo protein design are likely to support a broad range of applications, but larger complexes will be easier to create if a building block approach is adopted. Now protein filaments with tunable geometry can be made using assemblies that have both cyclic and superhelical symmetries aligned along the same axis.
- Jeremy R. H. Tame
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Research Briefing |
Phase-separation behaviour of RNAs
Ribonucleoprotein granules are ubiquitous in living organisms with the protein and RNA components having distinct roles. In the absence of proteins, RNAs are shown to undergo phase separation upon heating. This transition is driven by desolvation entropy and ion-mediated crosslinking and is tuned by the chemical specificity of the RNA nucleobases.
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Article |
RNAs undergo phase transitions with lower critical solution temperatures
The physicochemical driving forces of protein-free, RNA-driven phase transitions were previously unclear, but it is now shown that RNAs undergo entropically driven liquid–liquid phase separation upon heating in the presence of magnesium ions. In the condensed phase, RNAs can undergo an enthalpically favourable percolation transition that leads to arrested condensates.
- Gable M. Wadsworth
- , Walter J. Zahurancik
- & Priya R. Banerjee
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Article
| Open AccessThe interface of condensates of the hnRNPA1 low-complexity domain promotes formation of amyloid fibrils
Understanding of the molecular mechanisms underlying the maturation of protein condensates into amyloid fibrils associated with neurodegenerative diseases has so far remained elusive. Now it has been shown that in condensates formed by the low-complexity domain of the amyotrophic lateral sclerosis-associated protein hnRNPA1, fibril formation is promoted at the interface, which provides a potential therapeutic target for counteracting aberrant protein aggregation.
- Miriam Linsenmeier
- , Lenka Faltova
- & Paolo Arosio
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Research Briefing |
Visualization of protein motions using temperature-jump crystallography
An infrared laser-induced temperature jump provides a rapid and broadly applicable perturbation to protein dynamics. Temperature-jump crystallography was paired with time-resolved X-ray crystallography to study the dynamic enzyme lysozyme. Measurements with and without a functional inhibitor revealed different patterns in the propagation of motion throughout the enzyme.
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Article
| Open AccessMapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography
Shifts in temperature alter the structure and dynamics of macromolecules. Now, infra-red laser-induced temperature jump is combined with X-ray crystallography to observe protein structural dynamics in real time. Using this method, motions related to the catalytic cycle of lysozyme, a model enzyme, are visualized at atomic resolution and across broad timescales.
- Alexander M. Wolff
- , Eriko Nango
- & Michael C. Thompson
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Article
| Open AccessProtein–lipid charge interactions control the folding of outer membrane proteins into asymmetric membranes
Biological membranes are asymmetric bilayers, but little is known about how this asymmetry modulates membrane protein folding or stability. Now, folding and stability assays with bacterial outer membrane proteins reveal an exquisite sensitivity to asymmetric membrane charge distribution and a required matching of protein charge for efficient folding.
- Jonathan M. Machin
- , Antreas C. Kalli
- & Sheena E. Radford
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Article |
Kinetic control of shape deformations and membrane phase separation inside giant vesicles
The kinetics of liquid–liquid phase separation (LLPS) in cell-like confinements remains poorly understood. Now it has been shown that it involves complex interplay between the incipient phases and the membrane boundary, which arrests phase coarsening, deforms the membrane and couples LLPS with lipid phase separation.
- Wan-Chih Su
- , James C. S. Ho
- & Atul N. Parikh
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Article
| Open AccessDesign of allosteric sites into rotary motor V1-ATPase by restoring lost function of pseudo-active sites
Allostery produces concerted functions of protein complexes by orchestrating the cooperative work between the constituent subunits. By restoring functions of pseudo-active sites that have been lost through evolution, allosteric sites have now been designed into a rotary molecular motor, V1-ATPase, resulting in its rotation being boosted allosterically.
- Takahiro Kosugi
- , Tatsuya Iida
- & Nobuyasu Koga
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Article |
Mass photometric detection and quantification of nanoscale α-synuclein phase separation
The mechanism of α-synuclein amyloid aggregation via liquid–liquid phase separation has so far remained elusive. Now, the existence of nanoscale clusters of α-synuclein in sub-saturated concentrations is observed using mass photometry. These nanoscale clusters can act as precursors to both macroscopic condensate droplets as well as amyloid fibrils.
- Soumik Ray
- , Thomas O. Mason
- & Alexander K. Buell
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News & Views |
Polymeric protagonists for biological processes
Complexity is a hallmark of biological systems, but scientific experiments are typically conducted in simplified conditions. Now, diverse polymers that mimic the local environments of complex biological mixtures have been shown to improve protein folding, stability and function.
- Alana P. Gudinas
- & Danielle J. Mai
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Article
| Open AccessFibril formation and ordering of disordered FUS LC driven by hydrophobic interactions
Protein solutions can undergo liquid–liquid phase separation, by condensing into a dense phase that often resembles liquid droplets, which coexist with a dilute phase. Now it is shown that hydrophobic interactions, specifically at interfaces, can trigger a liquid–solid phase separation of a protein solution.
- Daria Maltseva
- , Sayantan Chatterjee
- & Mischa Bonn
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Article |
How synonymous mutations alter enzyme structure and function over long timescales
Enzymes with identical sequences of amino acids can display varying activities when encoded with mRNA with different properties, but why this is the case has been a mystery. Now, it has been shown that synonymous mutations in mRNA alter the partitioning of proteins into long-lived soluble misfolded states with varying activities.
- Yang Jiang
- , Syam Sundar Neti
- & Edward P. O’Brien
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Article
| Open AccessMass spectrometry captures biased signalling and allosteric modulation of a G-protein-coupled receptor
Native mass spectrometry has been used to interrogate both biased signalling and allosteric modulation of the β1-adrenergic receptor. Simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins has enabled the relative importance of specific interactions to be investigated.
- Hsin-Yung Yen
- , Idlir Liko
- & Carol V. Robinson
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Article |
Synthesis and single-molecule imaging reveal stereospecific enhancement of binding kinetics by the antitumour eEF1A antagonist SR-A3
The total synthesis and complete stereochemical assignment of the cyclic peptide natural product SR-A3—which has potential as a cancer therapeutic—has now been reported. Single-molecule biophysical and cellular experiments reveal a crucial, stereospecific role for a side-chain hydroxyl in SR-A3, which confers enhanced target residence time and efficacy in a mouse tumour model.
- Hao-Yuan Wang
- , Haojun Yang
- & Jack Taunton
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Perspective |
Challenges and opportunities in achieving the full potential of droplet interface bilayers
Droplet interface bilayers (DIBs) are a type of artificial bilayer that can act as cell membrane mimics. This Perspective surveys how DIBs can be used to mimic key cellular features (such as bilayer asymmetry) and processes (such as drug movement), and discusses challenges that need to be overcome to enable DIBs to reach their full potential as biomimetic model membranes.
- Elanna B. Stephenson
- , Jaime L. Korner
- & Katherine S. Elvira
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Article
| Open AccessFunctional DNA-based cytoskeletons for synthetic cells
Cytoskeletons are essential components of cells that perform a variety of tasks, and artificial cytoskeletons that perform these functions are required for the bottom-up assembly of synthetic cells. Now, a multi-functional cytoskeleton mimic has been engineered from DNA, consisting of confined DNA filaments that are capable of reversible self-assembly and transport of gold nanoparticles and vesicular cargo.
- Pengfei Zhan
- , Kevin Jahnke
- & Kerstin Göpfrich
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Article
| Open AccessUltrafast proton-coupled isomerization in the phototransformation of phytochrome
Phytochromes regulate plant growth by sensing far-red light through the photoisomerization of their protein-bound chromophores. In the phytochrome Agp2, it has now been demonstrated that ultrafast proton-transfer occurs from the chromophore to a protein–water network before photoisomerization, inducing protein changes on the ultrafast timescale. These protein changes develop further on longer timescales, resulting in an activated protein conformation.
- Yang Yang
- , Till Stensitzki
- & Karsten Heyne
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Article |
Single-particle combinatorial multiplexed liposome fusion mediated by DNA
Combinatorial high-throughput methodologies can accelerate screening and discovery in biochemistry and biomedical sciences, but they often rely on large-scale analyses, making them time-consuming and expensive. Now, DNA-mediated fusion of single liposomes has been shown to enable the spatially resolved and parallel cargo delivery of subattolitre volumes in a stochastic order of succession.
- Mette Galsgaard Malle
- , Philipp M. G. Löffler
- & Nikos S. Hatzakis
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Article
| Open AccessFrequency chasing of individual megadalton ions in an Orbitrap analyser improves precision of analysis in single-molecule mass spectrometry
The mass precision and resolution in charge-detection mass spectrometry can be improved by correcting frequency drifts of single ions. Now, chasing these individual ions for seconds in an Orbitrap mass spectrometer has revealed the exceptional stability of ultra-high-mass ions, culminating in an effective resolution of greater than 100,000 at m/z = 35,000.
- Tobias P. Wörner
- , Konstantin Aizikov
- & Albert J. R. Heck
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Article
| Open AccessCharge-density reduction promotes ribozyme activity in RNA–peptide coacervates via RNA fluidization and magnesium partitioning
Phase-separated compartments have long been proposed as precursors to cellular life. Now, it has been shown that RNA–peptide protocells are more robust when formed using shorter (rather than longer) peptides, and that peptide sequence determines the functional materials properties of these compartments.
- Juan M. Iglesias-Artola
- , Björn Drobot
- & Moritz Kreysing
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Article |
Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics
Coacervate microdroplets formed from pH- and redox-responsive peptides and self-assembled by liquid–liquid phase separation have been shown to quickly recruit macromolecular therapeutics—such as peptides, large proteins and mRNAs—and directly enter the cytosol of cells via a non-endocytic pathway. The subsequent release of therapeutic cargo is mediated by endogenic glutathione.
- Yue Sun
- , Sze Yi Lau
- & Ali Miserez
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News & Views |
Facing the fluctuations
Despite the disordered and dynamic environment in which it occurs, photosynthetic light harvesting is highly efficient. Now, measurements of energy transfer in single photosynthetic antennae show how these structures deal with protein fluctuations, robustly transferring the energy before it is lost.
- Pavel Malý
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Article |
Release of linker histone from the nucleosome driven by polyelectrolyte competition with a disordered protein
Histone H1 binds to nucleosomes with ultrahigh affinity, implying residence times incompatible with efficient biological regulation. Now it has been shown that the disordered regions of H1 retain their large-amplitude dynamics on the nucleosome, which enables a charged disordered histone chaperone to invade the H1–nucleosome complex and vastly accelerate H1 dissociation.
- Pétur O. Heidarsson
- , Davide Mercadante
- & Benjamin Schuler
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Article |
Observation of robust energy transfer in the photosynthetic protein allophycocyanin using single-molecule pump–probe spectroscopy
In photosynthesis, photoenergy transfers through chromophore-containing proteins, which exhibit thermal fluctuations that change the positions of the chromophores. Now the ultrafast dynamics in allophycocyanin—a cyanobacterial light-harvesting protein—have been measured using single-molecule pump–probe spectroscopy. The data show that energy transfer precedes protein-induced photophysical heterogeneity, ensuring that light harvesting is robust to the heterogeneity.
- Raymundo Moya
- , Audrey C. Norris
- & Gabriela S. Schlau-Cohen
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Article |
Deciphering how naturally occurring sequence features impact the phase behaviours of disordered prion-like domains
The complex link between protein sequence and phase behaviour for a family of prion-like low-complexity domains (PLCDs) has now been revealed. The results have uncovered a set of rules—which are interpreted using a stickers-and-spacers model—that govern the sequence-encoded phase behaviour of such PLCDs and enable physicochemical rationalizations that are connected to the underlying sequence composition.
- Anne Bremer
- , Mina Farag
- & Tanja Mittag
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Article |
Bottom-up fabrication of a proteasome–nanopore that unravels and processes single proteins
An integrated multiprotein nanopore has been fabricated using components from all three domains of life. This molecular machine opens the door to two approaches in single-molecule protein analysis, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then processed either as fragmented peptides or intact polypeptides.
- Shengli Zhang
- , Gang Huang
- & Giovanni Maglia
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Article |
A short peptide synthon for liquid–liquid phase separation
Liquid–liquid phase separation plays an important role in creating cellular compartments and protocells, but designing small-molecule models remains difficult. A peptide-based synthon for liquid–liquid phase separation consisting of two stickers and a flexible, polar spacer has now been presented. Condensates formed by these synthons can concentrate biomolecules and catalyse anabolic reactions.
- Manzar Abbas
- , Wojciech P. Lipiński
- & Evan Spruijt
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Article |
Heat flows in rock cracks naturally optimize salt compositions for ribozymes
The correct function of ribozymes in a prebiotic world would be dependent on the presence of optimal salt compositions and concentrations. Now, local heat fluxes have been shown to create an ideal salt habitat for ribozyme activity based on geologically plausible salt-leaching processes.
- T. Matreux
- , K. Le Vay
- & C. B. Mast
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Article |
A glycan gate controls opening of the SARS-CoV-2 spike protein
The opening mechanism of the SARS-CoV-2 spike protein has been studied by integrating computational and experimental data. Combining weighted ensemble molecular dynamics simulations, biolayer interferometry and ManifoldEM analysis of cryo-EM data revealed that the glycan at N343 plays a gating role in the opening mechanism of the SARS-CoV-2 spike protein.
- Terra Sztain
- , Surl-Hee Ahn
- & Rommie E. Amaro
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Article |
SARS-CoV-2 simulations go exascale to predict dramatic spike opening and cryptic pockets across the proteome
Simulations of the SARS-CoV-2 proteome that include over 0.1 s of aggregate data are reported. Spike opening was observed, revealing cryptic epitopes that differ between variants, explaining differential interactions with antibodies and receptors that determine pathogenicity. The cryptic pockets described provide new targets for antivirals and a wealth of mechanistic insight.
- Maxwell I. Zimmerman
- , Justin R. Porter
- & Gregory R. Bowman
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News & Views |
Finding the sweet spot for chaperone activity
Although critically important for protein function, post-translational modifications are complex and notoriously difficult to study. Now, the effects of O-GlcNAcylation on chaperone activity and the accompanying inhibition of amyloid fibril formation have been revealed, potentially yielding new routes to combat neurodegeneration.
- Sheena E. Radford
- & Theodoros K. Karamanos
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Article |
A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons
Voltage imaging is a powerful technique for studying electrical signalling in neurons. A palette of bright and sensitive voltage indicators has now been developed via enzyme-mediated ligation and Diels–Alder cycloaddition. Among these, a far-red indicator faithfully reports neuronal action potential dynamics with an excitation spectrum orthogonal to optogenetic actuators and green/red-emitting biosensors.
- Shuzhang Liu
- , Chang Lin
- & Peng Zou
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Article |
Sorting sub-150-nm liposomes of distinct sizes by DNA-brick-assisted centrifugation
Small liposomes of uniform sizes are valuable tools for studying membrane biology and developing drug-delivery vehicles. Now, a DNA-assisted sorting technique has been shown to produce multiple species of monodispersed liposomes with mean diameters below 150 nm in a scalable manner. This approach has enabled the high-resolution analyses of curvature-dependent membrane protein activities.
- Yang Yang
- , Zhenyong Wu
- & Chenxiang Lin
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Article |
Protein folding modulates the chemical reactivity of a Gram-positive adhesin
Bacteria use thioester-bond-containing proteins to covalently bind to host surfaces and withstand large mechanical shocks. Now, thioester bond reactivity has been shown to be force-dependent: forces >35 pN inhibit bond cleavage by primary amine ligands, whereas forces <6 pN enable reversible reformation. This force-modulated thioester bond reactivity could potentially enable bacterial mobility and a route by which they optimize infection.
- Alvaro Alonso-Caballero
- , Daniel J. Echelman
- & Julio M. Fernandez
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Article |
The catalytic dwell in ATPases is not crucial for movement against applied torque
Despite the fundamental role of ATPase in catalysing ATP hydrolysis, the structural and energetic aspects of this process are not fully understood. Coarse-grained computational models have now been used to calculate the free-energy surfaces of different types of ATPases. The catalytic dwell is shown not to be crucial for movement against applied torque.
- Chen Bai
- , Mojgan Asadi
- & Arieh Warshel
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Article |
De novo engineering of intracellular condensates using artificial disordered proteins
Artificial intrinsically disordered proteins (A-IDPs) have now been shown to form exclusionary, intracellular droplets that can be designed using simple principles that are based on the aromatic/aliphatic ratio and molecular weight. Droplets that sequester an enzyme and modulate enzyme efficiency on the basis of the molecular weight of the A-IDPs were also engineered using A-IDPs as a minimal condensate scaffold.
- Michael Dzuricky
- , Bradley A. Rogers
- & Ashutosh Chilkoti
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Article |
Barcoded DNA origami structures for multiplexed optimization and enrichment of DNA-based protein-binding cavities
Multivalent binding is a common strategy to enhance the interactions between weak binding partners. Now, following this principle, DNA origami scaffolds have been used to arrange DNA aptamers into specific geometries and to optimize linker spacings and flexibilities, which results in artificial binding sites with very high affinities for their corresponding ligands.
- Ali Aghebat Rafat
- , Sandra Sagredo
- & Friedrich C. Simmel
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Article |
α-Synuclein aggregation nucleates through liquid–liquid phase separation
The mechanism of nucleation for α-synuclein (α-Syn) aggregation and amyloid formation in Parkinson’s disease is unclear. Now, α-Syn has been shown to undergo liquid–liquid phase separation and a liquid-to-solid-like transition leading to amyloid fibril formation. This raises the possibility that liquid–liquid phase separation is a key pathogenic mechanism behind α-Syn aggregation in Parkinson’s disease.
- Soumik Ray
- , Nitu Singh
- & Samir K. Maji
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Article |
Dynamics of oligomer populations formed during the aggregation of Alzheimer’s Aβ42 peptide
Aβ42 oligomers are key toxic species associated with protein aggregation; however, the molecular pathways determining the dynamics of oligomer populations have remained unknown. Now, direct measurements of oligomer populations, coupled to theory and computer simulations, define and quantify the dynamics of Aβ42 oligomers formed during amyloid aggregation.
- Thomas C. T. Michaels
- , Andela Šarić
- & Tuomas P. J. Knowles
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Article |
Directional conformer exchange in dihydrofolate reductase revealed by single-molecule nanopore recordings
Single-molecule nanopore measurements have revealed ligand-induced conformational changes in the catalytic cycle of dihydrofolate reductase, and showed that the enzyme adopts distinctive conformers, which have different affinities for substrates and products. Crossing the transition state facilitates conformer exchange, suggesting that the chemical step catalyses the switch between conformers to obtain a more efficient product release.
- Nicole Stéphanie Galenkamp
- , Annemie Biesemans
- & Giovanni Maglia