Molecular biophysics

  • Article
    | Open Access

    m6A RNA post-transcriptional modification changes RNA hybridization kinetics. Here the authors show that the methylamino group can adopt syn-conformation pairing with uridine with a mismatch-like conformation in RNA duplex. They also develop a quantitative model that predicts how m6A affects the kinetics of hybridization.

    • Bei Liu
    • , Honglue Shi
    •  & Hashim M. Al-Hashimi
  • Article
    | Open Access

    F1Fo ATP synthase works using a rotary catalysis mechanism. Here, the authors report cryo-EM structures of Bacillus PS3 F1-ATPase encompassing the complete set of six states taken up during the catalytic cycle, including the binding- and catalytic-dwell states.

    • Meghna Sobti
    • , Hiroshi Ueno
    •  & Alastair G. Stewart
  • Article
    | Open Access

    Aβ oligomers (AβO) are thought to represent the main toxic species in Alzheimer’s disease but very high Aβ concentrations are required to study them in vitro and it remains unknown what role these off-pathway oligomers play in vivo. Here, the authors use a dimeric variant of Aβ termed dimAβ, where two Aβ40 units are linked, which facilitates to study AβO formation kinetics and they observe that Aβ off-pathway oligomer formation is strongly accelerated at endo-lysosomal pH, while amyloid fibril formation is delayed. Furthermore, the authors demonstrate that dimAβ is a disease-relevant model construct for pathogenic AβO formation by showing that dimAβ AβOs target dendritic spines and induce AD-like somatodendritic Tau missorting.

    • Marie P. Schützmann
    • , Filip Hasecke
    •  & Wolfgang Hoyer
  • Article
    | Open Access

    The nucleation mechanisms of biological protein phase separation are poorly understood. Here, the authors perform time-resolved SAXS experiments with the low-complexity domain (LCD) of hnRNPA1 and uncover multiple kinetic regimes on the micro- to millisecond timescale. Initially, individual proteins collapse. Nucleation then occurs via two steps distinguished by their protein cluster size distributions.

    • Erik W. Martin
    • , Tyler S. Harmon
    •  & Tanja Mittag
  • Article
    | Open Access

    Huntingtin exon-1 (HTTex1) consists of a N-terminal N17 domain, the disease causing polyQ domain and a C-terminal proline-rich domain (PRD). Here, the authors combine electron paramagnetic resonance (EPR), solid-state NMR with other biophysical method to characterise the structural differences of various HTTex1 fibril types with different toxicity and find that the dynamics and entanglement of the PRD domain differs among them and that the HTTex1 fibrils can be interconverted.

    • J. Mario Isas
    • , Nitin K. Pandey
    •  & Ansgar B. Siemer
  • Article
    | Open Access

    Ito and co-workers unravel how bacteria such as Salmonella switch gears with their flagellar driving machinery. External load triggers the dynamic remodeling of the molecular complex sustaining the torque, and the number of stator units is adapted in a non-trivial, cooperative manner.

    • Kenta I. Ito
    • , Shuichi Nakamura
    •  & Shoichi Toyabe
  • Article
    | Open Access

    During transcription, RNA polymerase II (RNAP2) is recruited to promoters and phosphorylated stepwise; so far, these steps have not been visualized in a single-copy gene in live cells. Here the authors use single-molecule microscopy to visualize endogenous phosphorylated RNAP2 and nascent mRNA synthesis at a single locus in living cells.

    • Linda S. Forero-Quintero
    • , William Raymond
    •  & Timothy J. Stasevich
  • Article
    | Open Access

    Artificial molecular systems can show complex kinetics of reproduction, however their integration into larger ensembles remains a challenge towards evolving higher order functionality. Here authors use show that self-reproducing lipids can initiate and accelerate octanol droplet movement and that reciprocally chemotactic movement of these droplets increases the rate of lipid reproduction substantially.

    • Dhanya Babu
    • , Robert J. H. Scanes
    •  & Nathalie Katsonis
  • Article
    | Open Access

    The primary energy conversion step in photosynthesis, charge separation, takes place in the reaction center. Here the authors investigate the heliobacterial reaction center using multispectral two-dimensional electronic spectroscopy, identifying the primary electron acceptor and revealing the charge separation mechanism.

    • Yin Song
    • , Riley Sechrist
    •  & Jennifer P. Ogilvie
  • Article
    | Open Access

    TMEM16 lipid scramblases transport lipids and also operate as ion channels with highly variable ion selectivities and various physiological functions. Using computational electrophysiology simulations, the authors identify the main ion-conductive state of TMEM16 lipid scramblases and find that lipid headgroups modulate ion permeability and regulate ion selectivity of TMEM16 proteolipidic pores.

    • Andrei Y. Kostritskii
    •  & Jan-Philipp Machtens
  • Article
    | Open Access

    Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their sensitivity and specificity are still poorly defined. Here, authors report a FRET-based sensor that informs directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level.

    • Janett Göhring
    • , Florian Kellner
    •  & Gerhard J. Schütz
  • Article
    | Open Access

    Photosystem II subunit S (PsbS) senses thylakoid lumen acidification when plants are exposed to excess light. Here the authors use NMR and IR spectroscopy to show that low pH causes repositioning of an amphipathic helix and folding of a loop involving critical pH sensing glutamate residues in PsbS.

    • Maithili Krishnan-Schmieden
    • , Patrick E. Konold
    •  & Anjali Pandit
  • Article
    | Open Access

    Chromosome segregation requires the association of the kinetochore protein complex with a specialized nucleosome at the centromere. Here, the authors present cryo-EM and mutational studies that provide insights into the structure of the budding yeast centromeric nucleosome and how the centromere CBF3 protein complex guides its formation.

    • Ruifang Guan
    • , Tengfei Lian
    •  & Yawen Bai
  • Article
    | Open Access

    Adeno-associated viruses (AAVs) have emerged as promising gene therapy vectors.The AAV capsid consists of 60 subunits made up from three distinct viral proteins (VPs). Here authors record high-resolution native mass spectra of intact AAV capsids to assess the VP stoichiometries in a panel of serotypes and reveals an extremely heterogeneous population of capsids of variable composition.

    • Tobias P. Wörner
    • , Antonette Bennett
    •  & Albert J. R. Heck
  • Article
    | Open Access

    An EGFR mutant with kinase domain duplication (EGFR-KDD) was previously identified in an index patient, but the functional and therapeutic implications remain unclear. Here, the authors show that KDD occurs in other ErbB receptors in multiple cancers, and characterize the mechanism and inhibition of EGFR-KDD.

    • Zhenfang Du
    • , Benjamin P. Brown
    •  & Christine M. Lovly
  • Article
    | Open Access

    Recent advances in computational methods have enabled the predictive design of self-assembling protein nanomaterials with atomic-level accuracy. Here authors investigate the assembly of two computationally designed, 120-subunit icosahedral complexes and find that assembly of each material from its two constituent protein building blocks was highly cooperative.

    • Adam J. Wargacki
    • , Tobias P. Wörner
    •  & Neil P. King
  • Article
    | Open Access

    The conversion of auditory and vestibular stimuli into electrical signals is initiated by force transmitted to a mechanotransduction channel through the tip link. Here authors show that a single tip-link bond is more mechanically stable relative to classic cadherins, and that the double stranded tip-link connection is stabilized by single strand rebinding facilitated by strong cis-dimerization domains.

    • Eric M. Mulhall
    • , Andrew Ward
    •  & Wesley P. Wong
  • Article
    | Open Access

    Spy is an ATP independent chaperone that can act as both a holdase and a foldase towards topologically simple substrates. Assessing the interaction of Spy and apoflavodoxin, a complex client, the authors show that Spy’s activity is substrate specific. Spy binds partially unfolded states of apoflavodoxin tightly, which limits the possibility of folding and converts Spy to a pure holdase.

    • Rishav Mitra
    • , Varun V. Gadkari
    •  & James C. A. Bardwell
  • Article
    | Open Access

    Liquid ribonucleoprotein condensates typically involve a dense network of multiple proteins and RNAs. Here, the authors employ a minimal system composed of Prion-like polypeptides (PLP), Arg-rich polypeptides (RRP), and RNA to form biphasic condensates with diverse morphologies tunable via mixture stoichiometry and hierarchy of intermolecular interactions.

    • Taranpreet Kaur
    • , Muralikrishna Raju
    •  & Priya R. Banerjee
  • Article
    | Open Access

    p23 is a co-chaperone of Hsp90 but its mode of action is mechanistically not well understood. Here, the authors combine in vitro and yeast in vivo assays, biochemical measurements and NMR experiments to characterize p23 and identify two conserved helical elements in the intrinsically disordered C-terminal tail of p23 that together with the folded domain of p23 regulate the Hsp90 ATPase activity and affect the binding and maturation of Hsp90 clients.

    • Maximilian M. Biebl
    • , Abraham Lopez
    •  & Johannes Buchner
  • Article
    | Open Access

    Our understanding of the molecular mechanisms underlying pathological protein aggregation remains incomplete. Here, single molecule infrared nanospectroscopy (AFM-IR) offers insight into the structure of Aβ42 oligomeric and fibrillar species and their interaction with an aggregation inhibitor, paving the way for single molecule drug discovery studies.

    • Francesco Simone Ruggeri
    • , Johnny Habchi
    •  & Tuomas P. J. Knowles
  • Article
    | Open Access

    Synaptic vesicle clusters were proposed to represent phase separated condensates. Here, the authors show that only two proteins, synapsin and synaptophysin, are sufficient to make vesicle clusters in fibroblasts which are similar to those found at synapses in morphology and liquid-like properties.

    • Daehun Park
    • , Yumei Wu
    •  & Sunghoe Chang
  • Article
    | Open Access

    Female honeybee larvae develop into queens when they are fed exclusively with royal jelly (RJ) and major royal jelly protein 1 (MRJP1) is the main protein component of RJ. Here, the authors present the 3.5 Å cryo-EM structure of native RJ filament, where MRJP1 forms the outer shell surrounding apisimin tetramers with bound lipids at the core of the filament.

    • Simone Mattei
    • , Arvid Ban
    •  & Daniel Boehringer
  • Article
    | Open Access

    Immune cells express immunoproteasomes (i20S), which bind to specialized regulators, contain different catalytic subunits and generate immunogenic peptides. HDX-MS—based assessment of the differences between the conformational dynamics of standard and i20s reveals specific, allosteric changes in i20S and upon regulator binding.

    • Jean Lesne
    • , Marie Locard-Paulet
    •  & Julien Marcoux
  • Article
    | Open Access

    The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology. Here, the authors use a combination of live cell imaging and in vitro single molecule binding measurements to show that tandem calponin homology domains (CH1–CH2) are sensitive to actin filament conformation, biasing their subcellular localization.

    • Andrew R. Harris
    • , Pamela Jreij
    •  & Daniel A. Fletcher
  • Article
    | Open Access

    For almost forty years, N-(1-pyrene) iodoacetamide has been used to label actin at C374, but the mechanisms of the fluorescence changes are still unknown due to the lack of structural information. Here authors provide cryo-EM structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP or ADP-phosphate in the active site.

    • Steven Z. Chou
    •  & Thomas D. Pollard
  • Article
    | Open Access

    Almost all allosteric systems are large multi-domain proteins which complicates the investigation of the mechanism in atomistic detail. Here authors designed a small allosteric protein system using the photocontrollable PDZ3 domain, that allows controlling allostery between the binding and allosteric site in both directions.

    • Olga Bozovic
    • , Brankica Jankovic
    •  & Peter Hamm
  • Article
    | Open Access

    The intrinsically disordered linker histone H1.0 and prothymosin α form a complex which exhibits slow exchange between bound and unbound populations at low protein concentrations and fast exchange at high concentrations. Here authors explain this observation by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange.

    • Andrea Sottini
    • , Alessandro Borgia
    •  & Benjamin Schuler
  • Article
    | Open Access

    The low-complexity (LC) domain mediates liquid-liquid phase separation and fibril formation of the RNA-binding protein FUS (FUsed in Sarcoma). Here, the authors combine cryo-EM, solid-state NMR measurements and MD simulations to structurally characterise the fibrils formed by the C-terminal half of the FUS LC domain and discuss stabilizing interactions within the fibril core.

    • Myungwoon Lee
    • , Ujjayini Ghosh
    •  & Robert Tycko
  • Article
    | Open Access

    The ribosome undergoes multiple large-scale structural rearrangements during protein elongation. Here the authors present an all-atom model of the ribosome to study the energetics of P/E hybrid-state formation, an early conformational rearrangement occurring during translocation.

    • Mariana Levi
    • , Kelsey Walak
    •  & Paul C. Whitford
  • Article
    | Open Access

    β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for catabolism of trehalose and maltose. Coupled analyses of multiple βPGM structures and enzymatic activity lead to the proposal of allomorphy — a post-translational mechanism controlling enzyme activity.

    • Henry P. Wood
    • , F. Aaron Cruz-Navarrete
    •  & Jonathan P. Waltho
  • Article
    | Open Access

    The xrRNA1 RNA from ZIKA virus (ZIKV) forms a complex ring-like architecture and is known for its mechanical anisotropy, but the mechanism for its direction-dependent mechanical responses remain unclear. Here authors use a single-molecule nanopore sensing technique combined with molecular dynamics simulations and show that the anisotropy in ZIKV xrRNA1 depends on Mg2+ and the key tertiary interactions.

    • Xiaolin Niu
    • , Qiuhan Liu
    •  & Xianyang Fang
  • Article
    | Open Access

    To overcome the limitation of FRET data being too sparse to cover all structural details, FRET experiments need to be carefully designed and complemented with simulations. Here the authors present a toolkit for automated design of FRET experiments, which determines how many and which FRET pairs should be used to maximize the accuracy, and for FRET-assisted structural modeling and refinement at the atomistic level.

    • Mykola Dimura
    • , Thomas-Otavio Peulen
    •  & Holger Gohlke
  • Article
    | Open Access

    GABAA receptors mediate most inhibitory synaptic transmission in the brain. Here authors used concatemeric α1β2γ2 GABAA receptors to introduce gain-of-desensitization mutations one subunit at a time, revealing non-concerted rearrangements with a key contribution of the γ2 subunit during desensitization.

    • Marc Gielen
    • , Nathalie Barilone
    •  & Pierre-Jean Corringer
  • Article
    | Open Access

    Cyanobacterial photosystem I has a highly conserved core antenna consisting of eleven subunits and more than 90 chlorophylls. Here via CryoEM and spectroscopy, the authors determine the location of a red-shifted low-energy chlorophyll that allows harvesting of longer wavelengths of light.

    • Hila Toporik
    • , Anton Khmelnitskiy
    •  & Yuval Mazor
  • Article
    | Open Access

    Determining molecular clustering in Photoactivated Localization Microscopy (PALM) experiments requires knowledge of the blinking properties of the fluorophore to prevent overcounting artefacts. Here the authors develop an experimental and analytical framework to determine the blinking parameters of fluorophores and incorporate this information into cluster analysis.

    • René Platzer
    • , Benedikt K. Rossboth
    •  & Mario Brameshuber
  • Article
    | Open Access

    SARS-CoV-2 spike protein binds host ACE2 for virus entry. Here, the authors determine kinetic and thermodynamic properties of this interaction using atomic force microscopy, develop peptides that inhibit binding and suggest existence of additional attachment factors.

    • Jinsung Yang
    • , Simon J. L. Petitjean
    •  & David Alsteens
  • Article
    | Open Access

    DNA glycosylases are lesion-specific enzymes that recognize specific nucleobase damages and catalyze their excision through cleavage of the glycosidic bond. Here, the authors present the crystal structures of human 8-oxoguanine (oxoG) DNA glycosylase bound to undamaged DNA and to DNA containing an intrahelical oxoG lesion and further analyse these structures with molecular dynamics simulations, which allows them to characterise the base-extrusion pathways.

    • Uddhav K. Shigdel
    • , Victor Ovchinnikov
    •  & Gregory L. Verdine
  • Article
    | Open Access

    Photoreceptor UVR8 in plants senses environmental UV levels through 26 structural tryptophan residues, but the role of 18 of them was unknown. The authors show, by experiments and computations, how these form a light-harvesting network that funnels the excitation to the pyramid centers enhancing the light-perception efficiency.

    • Xiankun Li
    • , Haisheng Ren
    •  & Dongping Zhong
  • Article
    | Open Access

    Protein structural dynamics can be studied by time-resolved crystallography (TRC) and ultrafast transient spectroscopic methods. Here, the authors perform electronic and vibrational transient absorption measurements to characterise the full photocycle of Photoactive Yellow Protein (PYP) both in the crystalline and solution state and find that the photocycle kinetics and structural intermediates of PYP deviate in the crystalline state, which must be taken into consideration when planning TRC experiments.

    • Patrick E. Konold
    • , Enis Arik
    •  & Marie Louise Groot
  • Article
    | Open Access

    Rescue of ribosomes stalled on non-stop mRNA is essential for cell viability, and several rescue systems to resolve stalling exist in bacteria. Here, the authors use rapid kinetics and cryo-EM to reveal the pathway and selectivity mechanism of ArfB-mediated ribosome rescue.

    • Kai-Hsin Chan
    • , Valentyn Petrychenko
    •  & Marina V. Rodnina
  • Article
    | Open Access

    Mevalonate diphosphate decarboxylase (MDD) is a key enzyme in the mevalonate pathway and catalyses the decarboxylation of mevalonate-5-diphosphate to isopentenyl diphosphate. Here, the authors provide insights into the conformational changes that occur during substrate binding of MDD and the subsequent enzymatic reaction steps by determining the substrate and intermediate bound crystal structures of Enterococcus faecalis MDD.

    • Chun-Liang Chen
    • , Lake N. Paul
    •  & Cynthia V. Stauffacher
  • Article
    | Open Access

    Human Histone Deacetylases (HDACs) regulate gene expression and are important drug targets. Here, the authors combine NMR measurements, enzymatic assays and molecular dynamics simulations and show that HDAC8 samples a catalytically active and an inactive state and further demonstrate that mutations and ligand binding alter the populations of the two states, which is of interest for inhibitor design.

    • Nicolas D. Werbeck
    • , Vaibhav Kumar Shukla
    •  & D. Flemming Hansen