Real-time observation of ligand-induced allosteric transitions in a PDZ domain.
Ontology highlight
ABSTRACT:
SUBMITTER: Bozovic O
PROVIDER: S-EPMC7585012 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Graphene films grown by vapour deposition tend to be polycrystalline due to the nucleation and growth of islands with different in-plane orientations. Here, using low-energy electron microscopy, we find that micron-sized graphene islands on Ir(111) rotate to a preferred orientation during thermal annealing. We observe three alignment mechanisms: the simultaneous growth of aligned domains and dissolution of rotated domains, that is, 'ripening'; domain boundary motion within islands; and continuous lattice rotation of entire domains. By measuring the relative growth velocity of domains during ripening, we estimate that the driving force for alignment is on the order of 0.1?meV per C atom and increases with rotation angle. A simple model of the orientation-dependent energy associated with the moiré corrugation of the graphene sheet due to local variations in the graphene-substrate interaction reproduces the results. This work suggests new strategies for improving the van der Waals epitaxy of 2D materials.
| S-EPMC4411305 | biostudies-literature
Project description:CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3' flap generated by NTS cleavage can be rapidly digested by a 3' to 5' single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3' flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9's conformation and suggest possible biotechnological applications.
| S-EPMC7812825 | biostudies-literature
Project description:The CRISPR-associated protein Cas9 is widely used for genome editing because it cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have revealed the multi-domain architecture of Cas9 and suggested sequential domain movements of Cas9 upon binding to the sgRNA and the target DNA These studies also hinted at the flexibility between domains; however, it remains unclear whether these flexible movements occur in solution. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET We found that the flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.
| S-EPMC5978321 | biostudies-literature
Project description:The transient receptor potential vanilloid type 1 (TRPV1) is a multimodal receptor which responds to various stimuli, including capsaicin, protons, and heat. Recent advances in cryo-electron microscopy have revealed the structures of TRPV1. However, due to the large size of TRPV1 and its structural complexity, the detailed process of channel gating has not been well documented. In this study, we applied the diffracted X-ray tracking (DXT) technique to analyze the intracellular domain dynamics of the TRPV1 protein. DXT enables the capture of intramolecular motion through the analysis of trajectories of Laue spots generated from attached gold nanocrystals. Diffraction data were recorded at two different frame rates: 100 μs/frame and 12.5 ms/frame. The data from the 100 μs/frame recording were further divided into two groups based on the moving speed, using the lifetime filtering technique, and they were analyzed separately. Capsaicin increased the slope angle of the MSD curve of the C-terminus in 100 μs/frame recording, which accompanied a shifting of the rotational bias toward the counterclockwise direction, as viewed from the cytoplasmic side. This capsaicin-induced fluctuation was not observed in the 12.5 ms/frame recording, indicating that it is a high-frequency fluctuation. An intrinsiccounterclockwise twisting motion was observed in various speed components at the N-terminus, regardless of the capsaicin administration. Additionally, the competitive inhibitor AMG9810 induced a clockwise twisting motion, which is the opposite direction to capsaicin. These findings contribute to our understanding of the activation mechanisms of the TRPV1 channel.
| S-EPMC10456751 | biostudies-literature
Project description:Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed BLInCR (Blue Light-Induced Chromatin Recruitment) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a reporter gene cluster in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ~2 minutes after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models for transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.
2017-11-22 | GSE103868 | GEO
Project description:Allostery represents a fundamental mechanism of biological regulation that is mediated via long-range communication between distant protein sites. Although little is known about the underlying dynamical process, recent time-resolved infrared spectroscopy experiments on a photoswitchable PDZ domain (PDZ2S) have indicated that the allosteric transition occurs on multiple timescales. Here, using extensive nonequilibrium molecular dynamics simulations, a time-dependent picture of the allosteric communication in PDZ2S is developed. The simulations reveal that allostery amounts to the propagation of structural and dynamical changes that are genuinely nonlinear and can occur in a nonlocal fashion. A dynamic network model is constructed that illustrates the hierarchy and exceeding structural heterogeneity of the process. In compelling agreement with experiment, three physically distinct phases of the time evolution are identified, describing elastic response ([Formula: see text] ns), inelastic reorganization ([Formula: see text] ns), and structural relaxation ([Formula: see text]s). Issues such as the similarity to downhill folding as well as the interpretation of allosteric pathways are discussed.
| S-EPMC5565459 | biostudies-literature
Project description:Domain patterns of perpendicularly magnetized ultra-thin ferromagnetic films are often determined by the competition of the short range but strong exchange interaction favouring ferromagnetic alignment of magnetic moments and the long range but weak antiferromagnetic dipolar interaction. Detailed phase diagrams of the resulting stripe domain patterns have been evaluated in recent years; however, the domain fluctuations in these pattern forming systems have not been studied in great detail so far. Here we show that domain fluctuations can be observed in ultra-thin two-dimensional ferromagnetic Fe/Ni/Cu(001) films with perpendicular magnetization in the stripe domain phase. Non-stroboscopic time-resolved threshold photoemission electron microscopy with high temporal resolution allows analysing the dynamic fingerprint of the topological excitations in the nematic domain phase. Furthermore, proliferation of domain ending defects in the vicinity of the spin reorientation transition is witnessed.
| S-EPMC4423231 | biostudies-literature
Project description:Conformational transitions play a central role in the phosphorylation mechanisms of protein kinase. To understand the nature of these transitions, we investigated the dynamics of nucleotide binding to the catalytic domain of PKA, a prototype for the protein kinase enzyme family. The open-to-closed transition in PKA was constructed as a function of ATP association by using available X-ray data and Brownian dynamics. Analyzing the multiple kinetic trajectories at the residue level, we find that the spatial rearrangement of the residues around the nucleotide-binding pocket, along with suppressed local fluctuations, controls the compaction of the entire molecule. In addition, to accommodate the stresses induced by ATP binding at the early transition stage, partial unfoldings (cracking) and reformations of several native contacts occur at the interfaces between the secondary structure motifs enveloping the binding pocket. This suggests that the enzyme experiences local structural deformations while reaching its functional, ATP-bound state. Our dynamical view of the ligand-induced transitions in PKA suggests that the kinetic hierarchy of local and global dynamics, the variable fluctuation of residues and the necessity of partial local unfolding may be fundamental components in other large scale allosteric transitions.
| S-EPMC2651249 | biostudies-literature
Project description:The mechanical stress due to shear flow has profound effects on cell proliferation, transport, gene expression, and apoptosis. The mechanisms for flow sensing and transduction are unclear, but it is postulated that fluid flow pulls upon the apical surface, and the resulting stress is eventually transmitted through the cytoskeleton to adhesion plaques on the basal surface. Here we report a direct observation of this flow-induced stress in the cytoskeleton in living cells using a parallel plate microfluidic chip with a fluorescence resonance energy transfer (FRET)-based mechanical stress sensor in actinin. The sensing cassette was genetically inserted into the cytoskeletal host protein and transfected into Madin-Darby canine kidney cells. A shear stress of 10 dyn/cm(2) resulted in a rapid increase in the FRET ratio indicating a decrease in stress across actinin with flow. The effect was reversible, and cells were able to respond to repeated stimulation and showed adaptive changes in the cytoskeleton. Flow-induced Ca(2+) elevation did not affect the response, suggesting that flow-induced changes in actinin stress are insensitive to intracellular Ca(2+) level. The reduction in FRET ratio suggests actin filaments are under normal compression in the presence of flow shear stress due to changes in cell shape, and/or actinin is not in series with actin. Treatment with cytochalasin-D that disrupts F-actin reduced prestress and the response to flow. The FRET/flow method is capable of resolving changes of stress in multiple proteins with optical spatial resolution and time resolution >1 Hz. This promises to provide insight into the force distribution and transduction in all cells.
| S-EPMC3174563 | biostudies-literature
Project description:Ever since its introduction by Ludwig Boltzmann, the ergodic hypothesis became a cornerstone analytical concept of equilibrium thermodynamics and complex dynamic processes. Examples of its relevance range from modeling decision-making processes in brain science to economic predictions. In condensed matter physics, ergodicity remains a concept largely investigated via theoretical and computational models. Here, we demonstrate the direct real-space observation of ergodicity transitions in a vertex-frustrated artificial spin ice. Using synchrotron-based photoemission electron microscopy we record thermally-driven moment fluctuations as a function of temperature, allowing us to directly observe transitions between ergodicity-breaking dynamics to system freezing, standing in contrast to simple trends observed for the temperature-dependent vertex populations, all while the entropy features arise as a function of temperature. These results highlight how a geometrically frustrated system, with thermodynamics strictly adhering to local ice-rule constraints, runs back-and-forth through periods of ergodicity-breaking dynamics. Ergodicity breaking and the emergence of memory is important for emergent computation, particularly in physical reservoir computing. Our work serves as further evidence of how fundamental laws of thermodynamics can be experimentally explored via real-space imaging.
| S-EPMC10499874 | biostudies-literature