Transient Intermediate States Research Articles

Optically Decoupling Electrochromic Dynamics and In Situ Morphological Evolution of a Single Soft Polyaniline Nanoentity.

Electroresponsive multicolored materials have tremendous potential in flexible electronics and smart wearable devices. Herein, the electrochromic dynamics and in situ morphological evolution of a single soft polyaniline nanoentity can be visualized and decoupled by an opto-electrochemical imaging strategy. The durability, tinting speed, and reversibility down to the single-nanoparticle level are quantified, and the switching of transient intermediate electrochromic states is trapped. The mechanistic studies suggest that the heterogeneity of electrochromic activity is attributed to the nonuniformity of the polymer network interspersed at the nanometric level. Furthermore, the representative Pauli repulsion effect is uncovered from the self-stretching behavior of the conductive state of polyaniline at the oxidized potential. It provides novel insights for advancing high-performance electrochromic devices and flexible strain sensors, which can be dynamically manipulated by external stimuli.

Xenon photoionization in the vicinity of 4d giant resonance and Cooper minimum using an XUV-NIR pump-probe experiment

The relaxation processes in atomic xenon following core ionization of the 4d and 4p subshells by extreme ultraviolet (XUV) pulses from a free-electron laser (FLASH) are investigated using ion time-of-flight spectroscopy. We compare the dynamics following ionization and Auger-Meitner decays at 90-eV photon energy, i.e., near the giant resonance, with those at 160 eV, near the Cooper minimum, where cross sections for photoionization of the 4d and 4p subshells are similar. Final states with charges higher than 4 show signatures of sequential absorption of two XUV photons, followed by subsequent Auger-Meitner decay. The averaged lifetimes of some important excited states are measured in a two-color XUV-pump–near-infrared-probe experiment. A transient enhancement in the ion yield of Xe5+ with an average lifetime of (49±3) fs is obtained, attributed to transient intermediate states following the decay of 4d double-core-hole states. Published by the American Physical Society 2024

Transient and elusive intermediate states in self‐assembly processes: An overview

AbstractThe transient and elusive intermediate states are the keys in self‐assembly processes, which are common phenomena shaping the structure, properties, and functionalities of assembled materials across many scientific domains. However, the understanding about the intermediate states of self‐assembly process is always challenging and limited. In this review, we focus on these states by combining theoretical and experimental approaches. By examining a wide variety of self‐assembly systems that span from biological to metal–organic nanostructures, this review uncovers the wealth of intermediate states of self‐assembled materials. In addition to combining the current knowledge, it will identify challenges and provide a new insight into the opportunities for future research.

Imaging biomacromolecules in action with liquid-phase electron microscopy

Imaging biomacromolecules in action in a liquid environment poses a challenge to single-molecule experiments. Liquid-phase electron microscopy (LP-EM) has demonstrated its unique ability to reveal previously unseen transient dynamics and intermediate states of delicate biomacromolecular systems (protein, DNA/RNA) with deep-learning methods and careful evaluation of electron beam effects.

Optical tracking of the heterogeneous solvent diffusion dynamics and swelling kinetics of single polymer microspheres.

The diffusion dynamics of small molecules into polymer entities is crucial for driving their morphology and function, which can be applied to research fields such as optical identification, medical implantation and intelligent sensing platforms. Herein, we demonstrate a nondestructive bright-field imaging strategy to monitor and control the morphology of polymer microspheres by varying the interfacial interaction and diffusion in a penetrant bath. The nanoscale interface movement of single polymer microspheres was tracked and converted into the diameter variation during the swelling event with sub-pixel accuracy, which is consistent with the calculation using Li-Tanaka's kinetic equations. More interestingly, the solvent diffusion dynamics along different directions of one particle are heterogeneous, indicating the non-uniform internal structure of a soft confined assembly. The swelling characteristics of single polymer microspheres can be quantified by this simple imaging strategy, and the transient intermediate swelling states are captured. To model the lifetime and stabilization times of microplastic entities, solvent selectivity and thermodynamic regulation were introduced to obtain the activation energy down to the single micro-entity level. This optical methodology shows capability for decoding the complex diffusion mechanism in polymer entities and provides guidance for the design of drug delivery systems, sensor platforms, and optical responsive materials.

The role of G protein conformation in receptor-G protein selectivity.

G protein-coupled receptors (GPCRs) selectively activate at least one of the four families of heterotrimeric G proteins, but the mechanism of coupling selectivity remains unclear. Structural studies emphasize structural complementarity of GPCRs and nucleotide-free G proteins, but selectivity is likely to be determined by transient intermediate-state complexes that exist before nucleotide release. Here we study coupling to nucleotide-decoupled G protein variants that can adopt conformations similar to receptor-bound G proteins without releasing nucleotide, and are therefore able to bypass intermediate-state complexes. We find that selectivity is degraded when nucleotide release is not required for GPCR-G protein complex formation, to the extent that most GPCRs interact with most nucleotide-decoupled G proteins. These findings demonstrate the absence of absolute structural incompatibility between noncognate receptor-G protein pairs, and are consistent with the hypothesis that transient intermediate states are partly responsible for coupling selectivity.

Surface Curvature Dominated Guest-Induced Nonequilibrium Deformations of Single Covalent Organic Framework-300 Particles.

Understanding the guest-induced dynamic deformation process of covalent organic frameworks (COFs) is vitally important to further increase their stimulus-response performances. Here we report on the dark-field microscopic (DFM) imaging approach to in situ monitor the guest-induced deformation evolution of individual COF-300 crystals in real time. We observe not only transient and nonequilibrium intermediate deformation states but also local surface curvature-driven diverse adsorption behaviours of single COF-300 particles for dichloromethane (DCM), undergoing one, two, and multiple expansion-contraction deformations as well as contraction-to-expansion transition. The surface curvature-dominated deformations are ascribed to the significant differences in the adsorption capacity for DCM at the curved tip and flat side regions, in which DCM can be adsorbed preferentially by curved tip regions of COF-300.

Surface Curvature Dominated Guest‐Induced Nonequilibrium Deformations of Single Covalent Organic Framework‐300 Particles

AbstractUnderstanding the guest‐induced dynamic deformation process of covalent organic frameworks (COFs) is vitally important to further increase their stimulus‐response performances. Here we report on the dark‐field microscopic (DFM) imaging approach to in situ monitor the guest‐induced deformation evolution of individual COF‐300 crystals in real time. We observe not only transient and nonequilibrium intermediate deformation states but also local surface curvature‐driven diverse adsorption behaviours of single COF‐300 particles for dichloromethane (DCM), undergoing one, two, and multiple expansion‐contraction deformations as well as contraction‐to‐expansion transition. The surface curvature‐dominated deformations are ascribed to the significant differences in the adsorption capacity for DCM at the curved tip and flat side regions, in which DCM can be adsorbed preferentially by curved tip regions of COF‐300.

Time-resolved solid state NMR of biomolecular processes with millisecond time resolution

We review recent efforts to develop and apply an experimental approach to the structural characterization of transient intermediate states in biomolecular processes that involve large changes in molecular conformation or assembly state. This approach depends on solid state nuclear magnetic resonance (ssNMR) measurements that are performed at very low temperatures, typically 25–30 K, with signal enhancements from dynamic nuclear polarization (DNP). This approach also involves novel technology for initiating the process of interest, either by rapid mixing of two solutions or by a rapid inverse temperature jump, and for rapid freezing to trap intermediate states. Initiation by rapid mixing or an inverse temperature jump can be accomplished in approximately-one millisecond. Freezing can be accomplished in approximately 100 microseconds. Thus, millisecond time resolution can be achieved. Recent applications to the process by which the biologically essential calcium sensor protein calmodulin forms a complex with one of its target proteins and the process by which the bee venom peptide melittin converts from an unstructured monomeric state to a helical, tetrameric state after a rapid change in pH or temperature are described briefly. Future applications of millisecond time-resolved ssNMR are also discussed briefly.

Intermediate-state imaging of electrical switching and quantum coupling of molybdenum disulfide monolayer

SignificanceThin transparent semiconductors of two-dimensional materials are attractive for the practical applications in next-generation nanoelectronic and optoelectronic devices. Probing the electron states and electrical switching mechanisms of a molybdenum disulphide monolayer with atomic-scale thickness (6.5 Å) allows us to unlock the full technological potential of this nanomaterial. We introduced a plasmonic phase imaging method to uncover the underlying mechanism and detailed switching dynamics of an electrical-state switching event. This dramatic phase change can be attributed to the reversible switching of classical electromagnetic coupling and quantum coupling effects interplaying between a single metal nanoparticle and molybdenum disulphide monolayer, and the transient intermediate states during the switching event can be directly imaged by a plasmonic technique.

A FRET-based method for monitoring structural transitions in protein self-organization

SummaryProteins assemble into a variety of dynamic and functional structures. Their structural transitions are often challenging to distinguish inside cells, particularly with a high spatiotemporal resolution. Here, we present a fluorescence resonance energy transfer (FRET)-based method for continuous and high-throughput monitoring of protein self-assemblies to reveal well-resolved transient intermediate states. Intermolecular FRET with both the donor and acceptor proteins at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We apply this method to monitor self-assembly of three proteins. We show that the mutant Huntingtin exon1 (mHttex1) first forms less-ordered assemblies, which develop into fibril-like aggregates, and demonstrate that the chaperone protein DNAJB6b increases the critical saturation concentration of mHttex1. We also monitor the structural changes in fused in sarcoma (FUS) condensates. This method adds to the toolbox for protein self-assembly structure and kinetics determination, and implementation with native or non-native proteins can inform studies involving protein condensation or aggregation.

Equilibria between conformational states of the Ras oncogene protein revealed by high pressure crystallography.

In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central regulatory processes occurring in multiple conformational states. Ras acts as a molecular switch between active GTP-bound, and inactive GDP-bound states, controlling essential signal transduction pathways. An allosteric network of interactions between the effector binding regions and the membrane interacting regions is involved in Ras cycling. The conformational states which coexist simultaneously in solution possess higher Gibbs free energy than the ground state. Equilibria between these states can be shifted by applying pressure favouring conformations with lower partial molar volume, and has been previously analyzed by high-pressure NMR spectroscopy. High-pressure macromolecular crystallography (HPMX) is a powerful tool perfectly complementary to high-pressure NMR, allowing characterization at the molecular level with a high resolution the different allosteric states involved in the Ras cycling. We observe a transition above 300 MPa in the crystal leading to more stable conformers. Thus, we compare the crystallographic structures of Ras(wt)·Mg2+·GppNHp and Ras(D33K)·Mg2+·GppNHp at various high hydrostatic pressures. This gives insight into per-residue descriptions of the structural plasticity involved in allosteric equilibria between conformers. We have mapped out at atomic resolution the different segments of Ras protein which remain in the ground-state conformation or undergo structural changes, adopting excited-energy conformations corresponding to transient intermediate states. Such in crystallo phase transitions induced by pressure open the possibility to finely explore the structural determinants related to switching between Ras allosteric sub-states without any mutations nor exogenous partners.

Time-resolved structural analysis of an RNA-cleaving DNA catalyst.

The 10-23 DNAzyme is one of the most prominent catalytically active DNA sequences1,2. Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential2. However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action3. Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10-23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme-RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of theDNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes.

HIV-Cell Membrane Fusion Intermediates are Restricted by Serinc3 and Serinc5

Serinc3 and Serinc5 are recently described host restriction factors that can block HIV infection by incorporating into budding viral particles and decreasing their ability to infect subsequent cells. Serincs are thought to block the very earliest stages of infection, membrane fusion and cell entry, by an incompletely understood mechanism. Understanding how Serincs disrupt HIV membrane fusion will clarify the requirements for normal HIV membrane fusion and could identify new viral weaknesses and drug targets. We used giant plasma membrane vesicles (blebs) as target membranes to study “wildtype” and Serinc-disrupted HIV membrane fusion at a single-particle level by fluorescence microscopy and cryo-Electron Tomography (cryoET). Using fluorescent reporters of membrane and content mixing, we observed that Serinc3 and Serinc5 cause no defect in mixing of the outer lipid leaflets (hemifusion), but a pronounced defect in fusion pore opening. Additionally, cryo-ET of HIV pseudoviruses mixed with blebs allowed observation of multiple intermediates of HIV membrane fusion. We found that Serinc3 and Serinc5 increased the number of hemifusion and early fusion product events observed in these tomograms and many of the fusion products have a narrowed neck between former virus and bleb. These results suggest that Serinc3 and Serinc5 restrict HIV membrane fusion, not by inhibiting a particular intermediate step, but by causing broad changes to the energetics of fusion that enables more frequent observation of multiple transient intermediate states.

Spectral Analysis of a Fast Biomolecular Transition in Magnetic Tweezers Measurements

Dynamic structural changes in biomolecules such as proteins and DNA are crucial for their correct function. These processes often involve transient intermediate states that occur on the (sub-)millisecond timescale. The investigation of such fast dynamics is experimentally challenging in particular in single molecule measurements. Here we investigate the limited branch migration of a Holliday junction within the torsionally constraint DNA of a magnetic tweezers setup. We gain access to the force-dependent stepping dynamics of the junction in the xx µs to x ms range using power spectral density analysis of the DNA length fluctuations. We expect that this technique can be extended to other biological systems where the individual structural transitions are too short and fast to be resolved in real-time. In our experiments the DNA length is obtained from the position of an attached magnetic particle. Our spectral analysis also reveals the rotational fluctuations of the magnetic particle due to an imperfect pinning in the magnetic field and its influence on the spatiotemporal resolution of magnetic tweezers.

Intermediate states of molecular self-assembly from liquid-cell electron microscopy

Traditional single-molecule methods do not report whole-molecule kinetic conformations, and their adaptive shape changes during the process of self-assembly. Here, using graphene liquid-cell electron microscopy with electrons of low energy at low dose, we show that this approach resolves the time dependence of conformational adaptations of macromolecules for times up to minutes, the resolution determined by motion blurring, with DNA as the test case. Single-stranded DNA molecules are observed in real time as they hybridize near the solid surface to form double-stranded helices; we contrast molecules the same length but differing in base-pair microstructure (random, blocky, and palindromic hairpin) whose key difference is that random sequences possess only one stable final state, but the others offer metastable intermediate structures. Hybridization is observed to couple with enhanced translational mobility and torsion-induced rotation of the molecule. Prevalent transient loops are observed in error-correction processes. Transient melting and other failed encounters are observed in the competitive binding of multiple single-stranded molecules. Among the intermediate states reported here, some were predicted but not observed previously, and the high incidence of looping and enhanced mobility come as surprises. The error-producing mechanisms, failed encounters, and transient intermediate states would not be easily resolved by traditional single-molecule methods. The methods generalize to visualize motions and interactions of other organic macromolecules.

Assembly of a GPCR-G Protein Complex

The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.

Insights into the Aggregation Mechanism of PolyQ Proteins with Different Glutamine Repeat Lengths

Polyglutamine (polyQ) diseases, including Huntington’s disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease’s onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states. We present an attenuated total reflection Fourier-transform infrared spectroscopic approach combined with protein immobilization to study polyQ aggregation dependent on the polyQ length. PolyQ proteins were engineered mimicking the mammalian N-terminus fragment of the Huntingtin protein and containing a polyQ sequence with the number of glutamines below (Q11), close to (Q38), and above (Q56) the disease threshold. A monolayer of the polyQ construct was chemically immobilized on the internal reflection element of the attenuated total reflection cell, and the aggregation was initiated via enzymatic cleavage. Structural changes of the polyQ sequence were monitored by time-resolved infrared difference spectroscopy. We observed faster aggregation kinetics for the longer sequences, and furthermore, we could distinguish β-structured intermediates for the different constructs, allowing us to propose aggregation mechanisms dependent on the repeat length. Q11 forms a β-structured aggregate by intermolecular interaction of stretched monomers, whereas Q38 and Q56 undergo conformational changes to various β-structured intermediates, including intramolecular β-sheets.

Conserved properties of dentate gyrus neurogenesis across postnatal development revealed by single-cell RNA sequencing.

The dentate gyrus of the hippocampus is a brain region in which neurogenesis persists into adulthood; however, the relationship between developmental and adult dentate gyrus neurogenesis has not been examined in detail. Here we used single-cell RNA sequencing to reveal the molecular dynamics and diversity of dentate gyrus cell types in perinatal, juvenile, and adult mice. We found distinct quiescent and proliferating progenitor cell types, linked by transient intermediate states to neuroblast stages and fully mature granule cells. We observed shifts in the molecular identity of quiescent and proliferating radial glia and granule cells during the postnatal period that were then maintained through adult stages. In contrast, intermediate progenitor cells, neuroblasts, and immature granule cells were nearly indistinguishable at all ages. These findings demonstrate the fundamental similarity of postnatal and adult neurogenesis in the hippocampus and pinpoint the early postnatal transformation of radial glia from embryonic progenitors to adult quiescent stem cells.

Effects of Collective Histone State Dynamics on Epigenetic Landscape and Kinetics of Cell Reprogramming.

Cell reprogramming is a process of transitions from differentiated to pluripotent cell states via transient intermediate states. Within the epigenetic landscape framework, such a process is regarded as a sequence of transitions among basins on the landscape; therefore, theoretical construction of a model landscape which exhibits experimentally consistent dynamics can provide clues to understanding epigenetic mechanism of reprogramming. We propose a minimal gene-network model of the landscape, in which each gene is regulated by an integrated mechanism of transcription-factor binding/unbinding and the collective chemical modification of histones. We show that the slow collective variation of many histones around each gene locus alters topology of the landscape and significantly affects transition dynamics between basins. Differentiation and reprogramming follow different transition pathways on the calculated landscape, which should be verified experimentally via single-cell pursuit of the reprogramming process. Effects of modulation in collective histone state kinetics on transition dynamics and pathway are examined in search for an efficient protocol of reprogramming.