Dynamic Structural Rearrangements Research Articles

Femtosecond Electron-Transfer Dynamics across the D2O/Cs+/Cu(111) Interface: The Impact of Hydrogen Bonding.

Hydrogen bonding is essential in electron-transfer processes at water-electrode interfaces. We study the impact of the H-bonding of water as a solvent molecule on real-time electron-transfer dynamics across a Cs+-Cu(111) ion-metal interface using femtosecond time-resolved two-photon photoelectron spectroscopy. We distinguish in the formed water-alkali aggregates two regimes below and above two water molecules per ion. Upon crossing the boundary of these regimes, the lifetime of the excess electron localized transiently at the Cs+ ion increases from 40 to 60 fs, which indicates a reduced alkali-metal interaction. Furthermore, the energy transferred to a dynamic structural rearrangement due to hydration is reduced from 0.3 to 0.2 eV concomitantly. These effects are a consequence of H-bonding in the water-water interaction and the beginning formation of a nanoscale water network. This finding is supported by real-space imaging of the solvatomers and vibrational frequency shifts of the OH stretching and bending modes calculated for these specific interfaces.

Mapping of long stretches of highly conserved sequences in over 6 million SARS-CoV-2 genomes.

We identified 11 conserved stretches in over 6.3 million SARS-CoV-2 genomes including all the major variants of concerns. Each conserved stretch is ≥100 nucleotides in length with ≥99.9% conservation at each nucleotide position. Interestingly, six of the eight conserved stretches in ORF1ab overlapped significantly with well-folded experimentally verified RNA secondary structures. Furthermore, two of the conserved stretches were mapped to regions within the S2-subunit that undergo dynamic structural rearrangements during viral fusion. In addition, the conserved stretches were significantly depleted for zinc-finger antiviral protein (ZAP) binding sites, which facilitated the recognition and degradation of viral RNA. These highly conserved stretches in the SARS-CoV-2 genome were poorly conserved at the nucleotide level among closely related β-coronaviruses, thus representing ideal targets for highly specific and discriminatory diagnostic assays. Our findings highlight the role of structural constraints at both RNA and protein levels that contribute to the sequence conservation of specific genomic regions in SARS-CoV-2.

Ultrahigh‐Capacity Rocksalt Cathodes Enabled by Cycling‐Activated Structural Changes

AbstractMn‐redox‐based oxides and oxyfluorides are considered the most promising earth‐abundant high‐energy cathode materials for next‐generation lithium‐ion batteries. While high capacities are obtained in high‐Mn content cathodes such as Li‐ and Mn‐rich layered and spinel‐type materials, local structure changes and structural distortions ( often lead to voltage fade, capacity decay, and impedance rise, resulting in unacceptable electrochemical performance upon cycling. In the present study, structural transformations that exploit the high capacity of Mn‐rich oxyfluorides while enabling stable cycling, in stark contrast to commonly observed structural changes that result in rapid performance degradation, are reported. It is shown that upon cycling of a cation‐disordered rocksalt (DRX) cathode (Li1.1Mn0.8Ti0.1O1.9F0.1, an ultrahigh capacity of ≈320 mAh g−1 (energy density of ≈900 Wh kg−1) can be obtained through dynamic structural rearrangements upon cycling , along with a unique voltage profile evolution and capacity rise. At high voltage, the presence of Mn4+ and Li+ vacancies promotes local cation ordering, leading to the formation of domains of a “δ phase” within the disordered framework. On deep discharge, Mn4+ reduction, along with Li+ insertion transform the structure to a partially ordered DRX phase with a β′‐LiFeO2‐type arrangement. At the nanoscale, domains of the in situ formed phases are randomly oriented, allowing highly reversible structural changes and stable electrochemical cycling. These new insights not only help explain the superior electrochemical performance of high‐Mn DRXbut also provide guidance for the future development of Mn‐based, high‐energy density oxide, and oxyfluoride cathode materials.

Time-resolved mechanisms of essential biological pathways.

The diversity of enzyme structure and function in living systems leads to exciting opportunities in biophysical research. Large numbers of these protein machines coordinate across space and time and undergo drastic changes in shape to carry out complex biological processes. My lab focuses on investigating precisely how genetic mutations impact the molecular function of key enzymes involved in DNA repair and/or RNA processing pathways. We use single molecule time-resolved fluorescence spectroscopy as a tool to explore in real time the protein-nucleic acid interactions and dynamic structural rearrangements that drive specific pathways. We are currently studying the detailed mechanisms SARS-CoV-2 viral RNA processing, ribosome assembly, and DNA mismatch repair. I will present recent results from pulsed interleaved excitation fluorescence resonance energy transfer (PIE-FRET) and fluorescence correlation spectroscopy (FCS) experiments. We aim to completely characterize the impact of mutations linked to disease on the molecular function of enzymes to understand how complex diseases develop at the molecular level and to identify new therapeutic targets.

Methane activation by vanadium oxide cluster anions (V2O5)NO- (N = 1-18).

The reactivity of vanadium oxide cluster anions (V2O5)NO- (N = 1-18) that feature with vanadium oxyl radicals (V-O⋅-) toward the most stable alkane, methane, at 273K has been characterized by employing a newly home-made ship-lock type reactor coupled with a time-of-flight mass spectrometer. The rate constants were determined in the orders of magnitude of 10-16-10-18cm3molecule-1s-1, which significantly breaks the detection limit of predecessors that the reactivity of metal-oxyl radicals (Mn+-O⋅-) with rate constants higher than 10-14cm3molecule-1s-1 could usually be measured. The dynamic structural rearrangement of the cluster skeleton has been proposed to account for the size-dependent reactivity of (V2O5)1-5O- clusters, which may also function in tuning the reactivity of large-sized (V2O5)6-18O- clusters. This work provides new insights into the mechanism of Mn+-O⋅--mediated C-H activation of methane at a strictly molecular level and expands the activity landscape of Mn+-O⋅- radicals.

Super-Resolution Microscopy for Structural Cell Biology.

Super-resolution microscopy techniques, and specifically single-molecule localization microscopy (SMLM), are approaching nanometer resolution inside cells and thus have great potential to complement structural biology techniques such as electron microscopy for structural cell biology. In this review, we introduce the different flavors of super-resolution microscopy, with a special emphasis on SMLM and MINFLUX (minimal photon flux). We summarize recent technical developments that pushed these localization-based techniques to structural scales and review the experimental conditions that are key to obtaining data of the highest quality. Furthermore, we give an overview of different analysis methods and highlight studies that used SMLM to gain structural insights into biologically relevant molecular machines. Ultimately, we give our perspective on what is needed to push the resolution of these techniques even further and to apply them to investigating dynamic structural rearrangements in living cells.

Phosphoinositide regulates dynamic movement of the S4 voltage sensor in the second repeat in two-pore channel 3

The two-pore channels (TPCs) are voltage-gated cation channels consisting of single polypeptides with two repeats of a canonical 6-transmembrane unit. TPCs are known to be regulated by various physiological signals such as membrane voltage and phosphoinositide (PI). The fourth helix in the second repeat (second S4) plays a major role in detecting membrane voltage, whereas the first repeat contains a PI binding site. Therefore, each of these stimuli is detected by a unique repeat to regulate the gating of the TPC central pore. How these various stimuli regulate the dynamic structural rearrangement of the TPC molecule remain unknown. Here, we found that PI binding to the first repeat in TPC3 regulates the movement of the distally located second S4 helix, showing that the PI-binding signal is not confined to the pore gate but also transmitted to the voltage sensor. Using voltage clamp fluorometry, measurement of gating charges, and Cys-accessibility analysis, we observed that PI binding significantly potentiates the voltage dependence of the movement of the second S4 helix. Notably, voltage clamp fluorometry analysis revealed that the voltage-dependent movement of the second S4 helix occurred in two phases, of which the second phase corresponds to the transfer of the gating charges. This movement was observed in the voltage range where gate-opening occurs and was potentiated by PI. In conclusion, this regulation of the second S4 helix by PI indicates a tight inter-repeat coupling within TPC3, a feature which might be conserved among TPC family members to integrate various physiological signals.

Markov State Models and Molecular Dynamics Simulations Provide Understanding of the Nucleotide-Dependent Dimerization-Based Activation of LRRK2 ROC Domain.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are recognized as the most frequent cause of Parkinson’s disease (PD). As a multidomain ROCO protein, LRRK2 is characterized by the presence of both a Ras-of-complex (ROC) GTPase domain and a kinase domain connected through the C-terminal of an ROC domain (COR). The bienzymatic ROC–COR–kinase catalytic triad indicated the potential role of GTPase domain in regulating kinase activity. However, as a functional GTPase, the detailed intrinsic regulation of the ROC activation cycle remains poorly understood. Here, combining extensive molecular dynamics simulations and Markov state models, we disclosed the dynamic structural rearrangement of ROC’s homodimer during nucleotide turnover. Our study revealed the coupling between dimerization extent and nucleotide-binding state, indicating a nucleotide-dependent dimerization-based activation scheme adopted by ROC GTPase. Furthermore, inspired by the well-known R1441C/G/H PD-relevant mutations within the ROC domain, we illuminated the potential allosteric molecular mechanism for its pathogenetic effects through enabling faster interconversion between inactive and active states, thus trapping ROC in a prolonged activated state, while the implicated allostery could provide further guidance for identification of regulatory allosteric pockets on the ROC complex. Our investigations illuminated the thermodynamics and kinetics of ROC homodimer during nucleotide-dependent activation for the first time and provided guidance for further exploiting ROC as therapeutic targets for controlling LRRK2 functionality in PD treatment.

Abstract OR-1: Condensed DNA Architecture in a Nucleoid of Escherichia coli

Background: Bacterial genomic DNA interacts with nucleoid-associated proteins (NAPs) and is located in a highly condensed and functional organized form in the nucleoid of the cell. The structure of the bacterial nucleoid is still awaiting its determination in high resolution. However, recent intensive research showed that condensed DNA in the bacterial nucleoid has a complex, hierarchically organized structure. Such architecture may only exist as a result of dynamic structural rearrangements, which characterize actively growing bacteria. Changes in environmental conditions are perceived by bacteria as stress. In the stationary phase caused by nutrient depletion, energy production processes become inefficient. Bacteria in the stationary phase use an energy-independent mechanism for maintaining an order to protect the DNA: the creation of stable structures, like those in inanimate nature. Cells develop into dormant forms that differ significantly in the structural organization from growing cells. Methods: Electron microscopy and synchrotron radiation diffraction studies were used to reveal distinct forms of DNA condensation in dormant E. coli cells. Results: The study made it possible to find the intracellular nanocrystalline, liquid crystalline, and folded nucleosome-like DNA structures, which were observed and described for the first time. Conclusion: The results of experiments made it possible to visualize the structures of the lower hierarchical tier of DNA compaction in the nucleoid of dormant cells. We hypothesized that the heterogeneity of bacterial cells allows for a flexible response to environmental changes and to surviving stress situations. Multiple types of DNA condensation in the same dormant E. coli cell increase the chances for rapid resumption of growth when conditions turn back to favorable.

Structural Consequences of Variation in SARS-CoV-2 B.1.1.7.

New globally circulating SARS-CoV-2 strains are causing concern about evolution of virus transmissibility, fitness and immune evasion mechanisms. A variant emerging from the United Kingdom called SARS-CoV-2 VUI 202012/01, or B.1.1.7, is thought to exhibit increased transmissibility that results from replication 4–10 times faster than the original Wuhan virus (Wuhan-Hu-1). Although this property is suspected to result from a specific mutation in the spike glycoprotein, D614G, there are 9 mutations that distinguish the UK variant B.1.1.7 from Wuhan-Hu-1 yet to be evaluated for functional effects. We asked if mutated positions fixed in UK variant B.1.1.7 may be involved in the virus life cycle, or evasion of the immune response, by modeling the UK variant spike protein and conducting structural analysis of mutations on the spike glycoprotein trimer (protomer) complexed to ACE2. Importantly, 4 out of 9 differences between the UK variant B.1.1.7 and Wuhan-Hu-1 spike protein alter direct intermolecular interactions. N501Y increased affinity between the spike protein and ACE2. The mutations at A570D, D614G and S982A reduced contact between individual chains of the trimeric spike protomer, potentially enhancing cleavage into S1 and S2 subunits, dynamic structural rearrangement and host cell fusion mechanisms. These data suggest that combined characteristics of mutations unique to UK variant B.1.1.7 enable high affinity binding to ACE2 and enhanced replication properties. The D614G mutation, associated with enhanced virus transmissibility, opens a potentially druggable structural pocket at the interface between spike glycoprotein subunits S1 and S2.

Direct energy transfer from photosystem II to photosystem I confers winter sustainability in Scots Pine

Evergreen conifers in boreal forests can survive extremely cold (freezing) temperatures during long dark winter and fully recover during summer. A phenomenon called “sustained quenching” putatively provides photoprotection and enables their survival, but its precise molecular and physiological mechanisms are not understood. To unveil them, here we have analyzed seasonal adjustment of the photosynthetic machinery of Scots pine (Pinus sylvestris) trees by monitoring multi-year changes in weather, chlorophyll fluorescence, chloroplast ultrastructure, and changes in pigment-protein composition. Analysis of Photosystem II and Photosystem I performance parameters indicate that highly dynamic structural and functional seasonal rearrangements of the photosynthetic apparatus occur. Although several mechanisms might contribute to ‘sustained quenching’ of winter/early spring pine needles, time-resolved fluorescence analysis shows that extreme down-regulation of photosystem II activity along with direct energy transfer from photosystem II to photosystem I play a major role. This mechanism is enabled by extensive thylakoid destacking allowing for the mixing of PSII with PSI complexes. These two linked phenomena play crucial roles in winter acclimation and protection.

Probing the Dynamic Structure-Function and Structure-Free Energy Relationships of the Coronavirus Main Protease with Biodynamics Theory.

The SARS-CoV-2 main protease (Mpro) is of major interest as an antiviral drug target. Structure-based virtual screening efforts, fueled by a growing list of apo and inhibitor-bound SARS-CoV/CoV-2 Mpro crystal structures, are underway in many laboratories. However, little is known about the dynamic enzyme mechanism, which is needed to inform both assay development and structure-based inhibitor design. Here, we apply biodynamics theory to characterize the structural dynamics of substrate-induced Mpro activation under nonequilibrium conditions. The catalytic cycle is governed by concerted dynamic structural rearrangements of domain 3 and the m-shaped loop (residues 132–147) on which Cys145 (comprising the thiolate nucleophile and half of the oxyanion hole) and Gly143 (comprising the second half of the oxyanion hole) reside. In particular, we observed the following: (1) Domain 3 undergoes dynamic rigid-body rotation about the domain 2–3 linker, alternately visiting two primary conformational states (denoted as M1pro ↔ M2pro); (2) The Gly143-containing crest of the m-shaped loop undergoes up and down translations caused by conformational changes within the rising stem of the loop (Lys137–Asn142) in response to domain 3 rotation and dimerization (denoted as M1/downpro ↔ 2·M2/uppro) (noting that the Cys145-containing crest is fixed in the up position). We propose that substrates associate to the M1/downpro state, which promotes the M2/downpro state, dimerization (denoted as 2·M2/uppro–substrate), and catalysis. Here, we explore the state transitions of Mpro under nonequilibrium conditions, the mechanisms by which they are powered, and the implications thereof for efficacious inhibition under in vivo conditions.

Babo1, formerly Vop1 and Cop1/2, is no eyespot photoreceptor but a basal body protein illuminating cell division in Volvox carteri.

In photosynthetic organisms many processes are light dependent and sensing of light requires light-sensitive proteins. The supposed eyespot photoreceptor protein Babo1 (formerly Vop1) has previously been classified as an opsin due to the capacity for binding retinal. Here, we analyze Babo1 and provide evidence that it is no opsin. Due to the localization at the basal bodies, the former Vop1 and Cop1/2 proteins were renamed V.c. Babo1 and C.r. Babo1. We reveal a large family of more than 60 Babo1-related proteins from a wide range of species. The detailed subcellular localization of fluorescence-tagged Babo1 shows that it accumulates at the basal apparatus. More precisely, it is located predominantly at the basal bodies and to a lesser extent at the four strands of rootlet microtubules. We trace Babo1 during basal body separation and cell division. Dynamic structural rearrangements of Babo1 particularly occur right before the first cell division. In four-celled embryos Babo1 was exclusively found at the oldest basal bodies of the embryo and on the corresponding d-roots. The unequal distribution of Babo1 in four-celled embryos could be an integral part of a geometrical system in early embryogenesis, which establishes the anterior-posterior polarity and influences the spatial arrangement of all embryonic structures and characteristics. Due to its retinal-binding capacity, Babo1 could also be responsible for the unequal distribution of retinoids, knowing that such concentration gradients of retinoids can be essential for the correct patterning during embryogenesis of more complex organisms. Thus, our findings push the Babo1 research in another direction.

Approaches to Exploring Spatio-Temporal Surface Dynamics in Nanoparticles with In Situ Transmission Electron Microscopy.

Many nanoparticles in fields such as heterogeneous catalysis undergo surface structural fluctuations during chemical reactions, which may control functionality. These dynamic structural changes may be ideally investigated with time-resolved in situ electron microscopy. We have explored approaches for extracting quantitative information from large time-resolved image data sets with a low signal to noise recorded with a direct electron detector on an aberration-corrected transmission electron microscope. We focus on quantitatively characterizing beam-induced dynamic structural rearrangements taking place on the surface of CeO2 (ceria). A 2D Gaussian fitting procedure is employed to determine the position and occupancy of each atomic column in the nanoparticle with a temporal resolution of 2.5 ms and a spatial precision of 0.25 Å. Local rapid lattice expansions/contractions and atomic migration were revealed to occur on the (100) surface, whereas (111) surfaces were relatively stable throughout the experiment. The application of this methodology to other materials will provide new insights into the behavior of nanoparticle surface reconstructions that were previously inaccessible using other methods, which will have important consequences for the understanding of dynamic structure-property relationships.

Dynamic structural rearrangements and functional regulation of voltage-sensing phosphatase.

The voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region. The latter contains a phosphatase domain and a C2 domain, showing remarkable similarity to the tumour suppressor enzyme PTEN. In VSP, membrane depolarization induces a conformational change in the VSD, which activates the phosphoinositide phosphatase. The final outcome in VSP is enzymatic activity in the cytoplasmic region, unlike in voltage-gated ion channels where conformational change of the transmembrane pore is induced by the VSD. Therefore, it is crucial to detect structural change in the cytoplasmic catalytic region to gain insights into the operating mechanisms of VSP. This review summarizes a recent study in which a method of genetic incorporation of a non-canonical amino acid, Anap, was used to detect dynamic membrane voltage-controlled rearrangements of the structure of the catalytic region of sea squirt VSP (Ci-VSP). Upon membrane depolarization, both the phosphatase domain and the C2 domain move in a similar time frame, suggesting that the two regions are coupled to each other. Measurement of Förster resonance energy transfer (FRET) between Anap introduced into the C2 domain of Ci-VSP and dipicrylamine in the cell membrane suggested no large movement of the enzyme towards the membrane. Fluorescence changes in Anap induced by different membrane potentials indicate the presence of multiple conformations of the active enzyme.

Ion Exchange Transformation of Magic-Sized Clusters

Ultrasmall semiconductor clusters are exciting materials because of their molecularly precise structures and their unique optical spectra. “Magic-sized” CdSe clusters are transformed into their Cu2Se counterparts by means of ion exchange. We leverage the molecularly precise structure and high sensitivity of these clusters to investigate the mechanism of cation exchange. We optically identify a metastable intermediate in the solid-state transformation. Isolation and characterization of this intermediate provide insight into the dynamic structural rearrangement of the cationic sublattice in the course of cation exchange and the role of ligand passivation. Such understanding of the dynamics of ion exchange at the solid–liquid interface could help engineer improved materials for solid-state electrolytes and energy storage devices.

Structural and Functional Characterization of Programmed Ribosomal Frameshift Signals in West Nile Virus Strains Reveals High Structural Plasticity Among cis-Acting RNA Elements

West Nile virus (WNV) is a prototypical emerging virus for which no effective therapeutics currently exist. WNV uses programmed -1 ribosomal frameshifting (-1 PRF) to synthesize the NS1' protein, a C terminally extended version of its non-structural protein 1, the expression of which enhances neuro-invasiveness and viral RNA abundance. Here, the NS1' frameshift signals derived from four WNV strains were investigated to better understand -1 PRF in this quasispecies. Sequences previously predicted to promote -1 PRF strongly promote this activity, but frameshifting was significantly more efficient upon inclusion of additional 3' sequence information. The observation of different rates of -1 PRF, and by inference differences in the expression of NS1', may account for the greater degrees of pathogenesis associated with specific WNV strains. Chemical modification and mutational analyses of the longer and shorter forms of the -1 PRF signals suggests dynamic structural rearrangements between tandem stem-loop and mRNA pseudoknot structures in two of the strains. A model is suggested in which this is employed as a molecular switch to fine tune the relative expression of structural to non-structural proteins during different phases of the viral replication cycle.

Wavelet-based multiscale analysis of bioimpedance data measured by electric cell-substrate impedance sensing for classification of cancerous and normal cells.

The paper presents a study to differentiate normal and cancerous cells using label-free bioimpedance signal measured by electric cell-substrate impedance sensing. The real-time-measured bioimpedance data of human breast cancer cells and human epithelial normal cells employs fluctuations of impedance value due to cellular micromotions resulting from dynamic structural rearrangement of membrane protrusions under nonagitated condition. Here, a wavelet-based multiscale quantitative analysis technique has been applied to analyze the fluctuations in bioimpedance. The study demonstrates a method to classify cancerous and normal cells from the signature of their impedance fluctuations. The fluctuations associated with cellular micromotion are quantified in terms of cellular energy, cellular power dissipation, and cellular moments. The cellular energy and power dissipation are found higher for cancerous cells associated with higher micromotions in cancer cells. The initial study suggests that proposed wavelet-based quantitative technique promises to be an effective method to analyze real-time bioimpedance signal for distinguishing cancer and normal cells.

The redox switch that regulates molecular chaperones.

Modification of reactive cysteine residues plays an integral role in redox-regulated reactions. Oxidation of thiolate anions to sulphenic acid can result in disulphide bond formation, or overoxidation to sulphonic acid, representing reversible and irreversible endpoints of cysteine oxidation, respectively. The antioxidant systems of the cell, including the thioredoxin and glutaredoxin systems, aim to prevent these higher and irreversible oxidation states. This is important as these redox transitions have numerous roles in regulating the structure/function relationship of proteins. Proteins with redox-active switches as described for peroxiredoxin (Prx) and protein disulphide isomerase (PDI) can undergo dynamic structural rearrangement resulting in a gain of function. For Prx, transition from cysteine sulphenic acid to sulphinic acid is described as an adaptive response during increased cellular stress causing Prx to form higher molecular weight aggregates, switching its role from antioxidant to molecular chaperone. Evidence in support of PDI as a redox-regulated chaperone is also gaining impetus, where oxidation of the redox-active CXXC regions causes a structural change, exposing its hydrophobic region, facilitating polypeptide folding. In this review, we will focus on these two chaperones that are directly regulated through thiol-disulphide exchange and detail how these redox-induced switches allow for dual activity. Moreover, we will introduce a new role for a metabolic protein, the branched-chain aminotransferase, and discuss how it shares common mechanistic features with these well-documented chaperones. Together, the physiological importance of the redox regulation of these proteins under pathological conditions such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis will be discussed to illustrate the impact and importance of correct folding and chaperone-mediated activity.

Novel Therapeutic Targets in Neuropsychiatric Disorders: The Neuroepigenome

The neuroepigenome, i.e., the epigenome of the nervous system, has become interesting for therapeutics in the last years due to widespread availability of dedicated drugs. A pivotal role for neuroepigenetics is certainly implied, both in physiology and pathology, by the highly dynamic structural and functional rearrangements that constantly occur into the nervous system, globally known as plasticity. Moreover, the idea that the pathophysiology of several neuropsychiatric disorders might involve epigenetic mechanisms is increasingly taking place due to accumulating experimental data and by the evidence of a synergistic interaction between genes and environment beneath most sporadic forms of these diseases. In this paper we will review the available evidence on the use of epigenome-modifying drugs in the field of neuropsychiatry, shortly describing for each disease the underlying assumptions of an epigenetic dysregulation.