Initial Folding Research Articles

Quasi-static large deformation compressive behaviour of origami-based metamaterials

An analytical model of the quasi-static response of Miura-ori based metamaterial to in-plane compression was first developed to describe the major mechanical characteristics related to the strength and energy absorption capacity of this material in the analysed loading direction. It is assumed that the base material is ductile and can be approximated as a perfectly plastic material. The results revealed that the initial strength and densification strain of the Miura-ori based metamaterial cannot be uniquely defined by its relative density, in contrast to most conventional cellular materials used for energy absorption applications. A particular value of the initial folding angle γ0 corresponds to the minimum relative density for each static sector angle α. This value determines the transition point separating the region where the initial material strength increases with the increase of the relative density from the one where this strength decreases with the increase of the relative density. Miura-ori based metamaterials with large initial dihedral angle γ0 have larger energy absorption capacity per unit mass, however exhibiting significant variations of Poisson's ratios during compression. The energy absorption efficiency of the Miura-ori based metamaterials was compared with that of single-walled regular honeycombs under in-plane compression. It is demonstrated that, for certain combination of the geometric parameters a, b, α and γ0, the origami-based materials can outperform the honeycombs with the same relative density. The analytical model was verified by the numerical simulations of the response of single cells with various geometries and multi-sheet origami configurations. The model predictions were also validated by a quasi-static compression test of a four-sheet origami specimen.

Prediction of folding mechanisms for Ig-like beta sandwich proteins based on inter-residue average distance statistics methods.

To understand the folding mechanism of a protein is one of the goals in bioinformatics study. Nowadays, it is enigmatic and difficult to extract folding information from amino acid sequence using standard bioinformatics techniques or even experimental protocols which can be time consuming. To overcome these problems, we aim to extract the initial folding unit for titin protein (Ig and fnIII domains) by means of inter-residue average distance statistics, Average Distance Map (ADM) and contact frequency analysis (F-value). TI I27 and TNfn3 domains are used to represent the Ig-domain and fnIII-domain, respectively. Beta-strands 2, 3, 5, and 6 are significant for the initial folding processes of TI I27. The central strands of TNfn3 were predicted as a primary folding segment. Known 3D structure and unknown 3D structure domains were investigated by structure or non-structure based multiple sequence alignment, respectively, to learn the conserved hydrophobic residues and predicted compact region relevant to evolution. Our results show good correspondence to experimental data, phi-value and protection factor from H-D exchange experiments. The significance of conserved hydrophobic residues near F-value peaks for structural stability using hydrophobic packing is confirmed. Our prediction methods once again could extract a folding mechanism only knowing the amino acid sequence.

Effects of Preferential Counterion Interactions on the Specificity of RNA Folding.

The real-time search for native RNA structure is essential for the operation of regulatory RNAs. We previously reported that a fraction of the Azoarcus ribozyme achieves a compact structure in less than a millisecond. To scrutinize the forces that drive initial folding steps, we used time-resolved SAXS to compare the folding dynamics of this ribozyme in thermodynamically isostable concentrations of different counterions. The results show that the size of the fast-folding population increases with the number of available counterions and correlates with the flexibility of initial RNA structures. Within 1 ms of folding, Mg2+ exhibits a smaller preferential interaction coefficient per charge, ΔΓ+/ Z, than Na+ or [Co(NH3)6]3+. The lower ΔΓ+/ Z corresponds to a smaller yield of folded RNA, although Mg2+ stabilizes native RNA more efficiently than other ions at equilibrium. These results suggest that strong Mg2+-RNA interactions impede the search for globally native structure during early folding stages.

Synthesis of Single-Ring Nanoparticles Mimicking Natural Cyclotides by a Stepwise Folding-Activation-Collapse Process.

Cyclotides are small cyclic polypeptides found in a variety of organisms, ranging from bacteria to plants. Their ring structure endows those polypeptides with specific properties, such as improved stability against enzymatic degradation. Optimal cyclotide activity is often observed only in the presence of intra-ring disulfide bonds. Synthesis of soft nano-objects mimicking the conformation of natural cyclotides remains challenging. Here, a new class of natural cyclotide mimics synthesized by a stepwise folding-activation-collapse process at high dilution starting from simple synthetic precursor polymers is established. The initial folding step is carried out by a photoactivated hetero Diels-Alder (HDA) ring-closing reaction, which is accompanied by chain compaction of the individual precursor polymer chains as determined by size exclusion chromatography (SEC). The subsequent activation step comprises a simple azidation procedure, whereas the final collapse step is driven by CuAAC in the presence of an external cross-linker, providing additional compaction to the final single-ring nanoparticles (SRNPs). The unique structure and compaction degree of the SRNPs is established via a detailed comparison with conventional single-chain nanoparticles (SCNPs) prepared exclusively by chain collapse from the exact same precursor polymer (without the prefolding step). The stepwise folding-activation-collapse approach opens new avenues for the preparation of artificial cyclotide mimetics.

Optimization of Initial Folding Square Sections for the Crashworthiness Design

In this study, crash behavior of optimization of initial folding square sections are evaluated. Three parameters of design are set as width (w), height (h) and thickness (t) with three levels. Deformation mode of crash box and energy absorbing due to frontal load is observed. Response surface method is used to optimize the Initial folding square sections. Box-Bhenken is chosen to provide this setting and 13 models is built. Computer simulation is used to determine the energy absorption by using Software Finite Element Analysis. Based on computer simulation results, it can be denoted that w =1.1 mm, h = 0.9 mm and t = 2.75 mm produce the highest of energy absorption.

Emplacement of the Rocche Rosse rhyolite lava flow (Lipari, Aeolian Islands)

The Rocche Rosse lava flow marks the most recent rhyolitic extrusion on Lipari island (Italy), and preserves evidence for a multi-stage emplacement history. Due to the viscous nature of the advancing lava (108 to 1010 Pa s), indicators of complex emplacement processes are preserved in the final flow. This study focuses on structural mapping of the flow to highlight the interplay of cooling, crust formation and underlying slope in the development of rhyolitic lavas. The flow is made up of two prominent lobes, small (< 0.2 m) to large (> 0.2 m) scale folding and a channelled geometry. Foliations dip at 2–4° over the flatter topography close to the vent, and up to 30–50° over steeper mid-flow topography. Brittle faults, tension gashes and conjugate fractures are also evident across flow. Heterogeneous deformation is evident through increasing fold asymmetry from the vent due to downflow cooling and stagnation. A steeper underlying topography mid-flow led to development of a channelled morphology, and compression at topographic breaks resulted in fold superimposition in the channel. We propose an emplacement history that involved the evolution through five stages, each associated with the following flow regimes: (1) initial extrusion, crustal development and small scale folding; (2) extensional strain, stretching lineations and channel development over steeper topography; (3) compression at topographic break, autobrecciation, lobe development and medium scale folding; (4) progressive deformation with stagnation, large-scale folding and re-folding; and (5) brittle deformation following flow termination. The complex array of structural elements observed within the Rocche Rosse lava flow facilitates comparisons to be made with actively deforming rhyolitic lava flows at the Chilean volcanoes of Chaitén and Cordón Caulle, offering a fluid dynamic and structural framework within which to evaluate our data.

Endoplasmic reticulum chaperone glucose-regulated protein 78 in gastric cancer: An emerging biomarker.

The endoplasmic reticulum (ER) is the principal organelle responsible for the synthesis, initial post-translational modification, folding, export and secretion of proteins. It is also responsible for the maintenance of cellular homeostasis. In response to cellular stress conditions including glucose deprivation, hypoxia and changes in calcium homeostasis, ER stress machinery is activated and triggers the unfolded protein response, resulting in the restoration of homeostasis or activation of cell death. Glucose-regulated protein 78 (GRP78), a molecular chaperone, may be induced by ER stress at the transcriptional and translational level. A number of studies have demonstrated that GRP78 serves an important role in tumor cell proliferation, metastasis, angiogenesis and drug-resistance. The present review systematically describes the association between GRP78 expression and gastric cancer pathogenesis, and emphasizes that GRP78 is a novel diagnostic and therapeutic biomarker of gastric cancer.

Systematic optimization of cell-free synthesized human endothelin B receptor folding.

Cell-free production of G-protein coupled receptors is becoming attractive for biochemical characterization, ligand screening or even structural purposes. However, despite high production levels within the range of mg/mL, the fraction of functionally folded receptor is frequently below 1%. In synthetic cell-free reactions, numerous factors that affect the efficient folding and stability of translated membrane proteins can be addressed by the appropriate design of the synthetic expression environment. We demonstrate the systematic quality optimization of the cell-free synthesized human endothelin B receptor by a combined approach of lipid screening, redox optimization, and molecular engineering. Key parameters for receptor folding are the implementation of nanodiscs, the selection of suitable lipid environments for co-translational solubilization, as well as providing an optimized redox system for essential disulfide bridge formation. In addition, enrichment with chaperones as well as receptor engineering by thermostabilization further supported the folding into ligand binding conformation. In summary, we provide evidence that the initial co-translational folding process rather than long-term stability of the receptor is limiting. The folding efficiency could be improved by more than 103-fold and under optimized conditions, up to 1.6 nmol or ∼100 µg of ligand binding competent receptor could be produced per mL of reaction mixture in a timescale of less than 24 h. The identified parameters affect rather common characteristics of G-protein receptors and are thus likely to improve the folding of similar targets as well. The optimized process provides full-length receptors embedded in defined membrane environments and in quantities and quality sufficient for throughput screening applications.

An Electromagnetic Tweezers for Studying Fast Protein Folding Dynamics

Protein folding under force does mechanical work, however, for that work to be physiologically relevant, it must be delivered fast enough to compete with the speed of molecular motors. For instance, it is not known if titin domains can fold fast enough to follow the myosin motors, thus contributing to the mechanical power during muscle contraction. Accurate measurement of the initial folding velocity requires applying fast force changes in order to resolve the initial folding events. Yet, most force-spectroscopy instruments change the force by moving mechanical components, introducing a time lag-at least 100 ms in the case of magnetic tweezers-where these early molecular events are lost. Here, we present a newly developed electromagnetic tweezers instrument, where the magnetic field is generated by an electromagnetic head. The force acting on the superparamagnetic beads depends on the electric current flowing through the head, and can therefore be changed as fast as current can be supplied. Based on Karlqvist approximation of the field created by an electromagnetic head, we work out a closed analytical expression that relates the force on the superparamagnetic beads with the electric current and the vertical distance of the head to the bead. We calibrate our instrument by relating this force expression with the step-sizes of protein L unfolding/refolding events, allowing to reach forces of 50 pN in 5 ms, when working at a distance of 200 μm and an electric current of 600 mA. This force range can potentially be increased up to 500 pN by using thinner fluid chambers, approaching the AFM force range, but with the exquisite stability of magnetic tweezers. Our instrument development offers a unique tool for studying how proteins respond mechanically to force perturbations as those experienced in vivo.

Non-Native α-Helices in the Initial Folding Intermediate Facilitate the Ordered Assembly of the β-Barrel in β-Lactoglobulin.

The roles of non-native α-helices frequently observed in the initial folding stage of β-sheet proteins have been examined for many years. We herein investigated the residue-level structures of several mutants of bovine β-lactoglobulin (βLG) in quenched-flow pH-pulse labeling experiments. βLG assumes a collapsed intermediate with a non-native α-helical structure (I0) in the early stage of folding, although its native form is predominantly composed of β-structures. The protection profile in I0 of pseudo-wild type (WT*) βLG was found to deviate from the pattern of the "average area buried upon folding" (AABUF). In particular, the level of protection at the region of strand A, at which non-native α-helices form in the I0 state, was significantly low compared to AABUF. G17E, the mutant with an increased helical propensity, showed a similar protection pattern. In contrast, the protection pattern for I0 of E44L, the mutant with an increased β-sheet propensity, was distinct from that of WT* and resembled the AABUF pattern. Transverse relaxation measurements demonstrated that the positions of the residual structures in the unfolded states of these mutants were consistent with those of the protected residues in the respective I0 states. On the basis of the slower conversion of I0 to the native state for E44L to that for WT*, non-native α-helices facilitate the ordered assembly of the β-barrel by preventing interactions that trap folding.

Identification of Key Interactions in the Initial Self-Assembly of Amylin in a Membrane Environment

Islet amyloid polypeptide, also known as amylin, forms aggregates that reduce the amount of insulin-producing cells in patients with type II diabetes mellitus. Much remains unknown about the process of aggregation and cytotoxicity, but it is known that certain cell membrane components can alter the rate of aggregation. Using atomistic molecular dynamics simulations combined with the highly mobile membrane mimetic model incorporating enhanced sampling of lipid diffusion, we investigate interaction of amylin peptides with the membrane components as well as the self-assembly of amylin. Consistent with experimental evidence, we find that an initial membrane-bound α-helical state folds into stable β-sheet structures upon self-assembly. Our results suggest the following mechanism for the initial phase of amylin self-assembly. The peptides move around on the membrane with the positively charged N-terminus interacting with the negatively charged lipid headgroups. When the peptides start to interact, they partly unfold and break some of the contacts with the membrane. The initial interactions between the peptides are dominated by aromatic and hydrophobic interactions. Oligomers are formed showing both intra- and interpeptide β-sheets, initially with interactions mainly in the C-terminal domain of the peptides. Decreasing the pH to 5.5 is known to inhibit amyloid formation. At low pH, His18 is protonated, adding a fourth positive charge at the peptide. With His18 protonated, no oligomerization is observed in the simulations. The additional charge gives a strong midpoint anchoring of the peptides to negatively charged membrane components, and the peptides experience additional interpeptide repulsion, thereby preventing interactions.

Linking pH, Temperature, and K+ Concentration for DNA i-Motif Formation.

The conformation a particular DNA segment assumes depends upon its sequence context and the environment under which it is prepared. To complement our findings with G-rich sequences related to the human telomere, we have been investigating the pH induced transition from single strand to i-motif for sequences related to the human telomere C-rich strand. We have carried out titrations of (CCCTAA)4 from pH 7.0 to pH 5.0 at temperatures ranging from 15 to 45 °C at 115 mM K+ and at K+ concentrations ranging from 15 to 215 mM at 25 °C. Circular dichroism (CD) spectra were determined to monitor the transition. The pH at the midpoint of the proton induced transition, pHmp, is dependent upon both temperature and [K+]. Wyman-type plots of log K vs pH yielded linear correlations and the slopes of those lines, ΔQ, were also linearly dependent on [K+] and T. For these studies, ΔQ represents the minimum number of protons that must be added to the oligomer to induce the initial folding. These results are consistent with Le Chatelier's principle. Optical melting studies were also carried out for (CCCTAA)4 at pH 5.0 and [K+] ranging from 15 to 315 mM. Linear correlations between the temperature at the midpoint of the transition, Tm, and log [K+] allowed determination of the differential ion binding term, ΔnK+. These linkages between pH, temperature, and [K+] can be utilized to design i-motif forming DNA oligomers with highly tunable properties.

Foam-Filled Grooved Tubes with Circular Cross Section Under Axial Compression: An Experimental Study

In this article, absorbed energy per unit of initial tube length, initial peak force and mean folding force in empty and polyurethane foam-filled simple and grooved specimens with circular cross section are investigated, experimentally. Results demonstrate that in the empty and foam-filled simple specimens, absorbed energy per unit of initial tube length, initial peak force and mean folding force are larger than the corresponding values of the empty and foam-filled grooved specimens, respectively. But, increment of absorbed energy due to the foam-filler in the grooved tubes is more than the corresponding value in the simple specimens. Also, diagrams of absorbed energy and folding force versus the grooves distance have the minimum value. Therefore, there is a critical value for the grooves distance and according to the application viewpoint, to produce a good energy absorber, the grooves distance is selected far from the critical grooves distance. The experimental results show that when the grooves distance (λ) decreases, deformation mode inclines to the concertina (regular) mode and when λ increases, the deformation mode inclines to the diamond mode. Finally, a semiempirical relation is derived to predict the initial peak force of empty and foam-filled grooved tubes.

The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling.

Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two α-helices connected by an α-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50-55 Å of the exit tunnel. The structure reveals how α-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome.

Translation and folding of single proteins in real time

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide's vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein's amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein's amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.

The nonlinear aeroelastic characteristics of a folding wing with cubic stiffness

This paper focuses on the nonlinear aeroelastic characteristics of a folding wing in the quasi-steady condition (namely at fixed folding angles) and during the morphing process. The structure model of the folding wing is formulated by the Lagrange equations, and the constraint equation is used to describe the morphing strategy. The aerodynamic influence coefficient matrices at several folding angles are calculated by the Doublet Lattice method, and described as rational functions in the Laplace domain by the rational function approximation, and then the Kriging agent model technique is adopted to interpolate the coefficient matrices of the rational functions, and the aerodynamics model of the folding wing during the morphing process is built. The aeroelastic responses of the folding wing with cubic stiffness are simulated, and the results show that the motion types of aeroelastic responses in the quasi-steady condition and during the morphing process are all sensitive to the initial condition and folding angle. During the morphing process, the transition of the motion types is observed. And apart from the period of transition, the aeroelastic response at some folding angles may exhibit different motion types, which can be found from the results in the quasi-steady condition.

Fault-related folds in the southern Pyrenees

ABSTRACT The fault-related folds observed in the south Pyrenean fold and thrust belt are hybrid structures with elements of detachment folding as well as elements of fault bend and fault propagation folding. Fanning growth strata in all the studied case studies indicate progressive limb rotation during deformation, mainly at their initial stages when fold growing mostly controlled the dispersal of syntectonic sediments as a result of higher uplift rates with respect to sedimentation rates. Break thrusts developed in the fold limb of the initial detachment folds to form fault propagation and fault bend geometries. Inversion folds developed at the rift margins. There the geometry of the folds as well as the brittle deformation patterns are controlled by the inherited fault systems, which are not symmetrical with respect to the fold axis. In the absence of previous structures, brittle patterns developed before or at the initial stages of folding by layer-parallel shortening preserving a symmetrical arrangement with respect to the fold axis.

Dynamic Lipid-dependent Modulation of Protein Topology by Post-translational Phosphorylation

Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.

Detection of Folding Sites of β-Trefoil Fold Proteins Based on Amino Acid Sequence Analyses and Structure-Based Sequence Alignment

The information on the 3D structure of a protein including its folding mechanism is encoded in its amino acid sequence. A β-trefoil protein is well known to have a remarkable 3D structure property, that is, the pseudo three-fold symmetry without clear hydrophobic packing. It is interesting to investigate how information on the folding mechanism to form such a topology is encoded in the amino acid sequence of a protein. In this study, analyses based on inter-residue average distance statistics and the conservation of hydrophobic residues are performed for sequences of 26 β-trefoil proteins to identify significant sites for the initial folding. Results are compared with the native 3D structures. The conserved hydrophobic residues are defined by multiple sequence alignment based on the 3D structures. It is confirmed that a conserved hydrophobic residue is always located in a β-strand. In particular, β-strands 5 and 6 are significant for the initial folding from the analyses based on the inter-residue average distance statistics. These results coincide well with the experimental data obtained so far for folding of some of the β-trefoil proteins. It is also confirmed that the conserved hydrophobic residues defined in this study contribute to form hydrophobic packing in β-trefoil proteins in general. Twelve conserved hydrophobic residue pairs are almost always observed to form packing in the 26 β-trefoil proteins from different superfamilies. We elucidate how the conserved hydrophobic residues in β-strands 5 and 6 contribute to the initial stage of folding of a β-trefoil protein. The common packing of the 12 conserved hydrophobic residue pairs are significant to form the whole β-trefoil fold structure.

Geomechanical Modeling of Stress and Fracture Distribution during Contractional Fault-Related Folding

Understanding and predicting the distribution of fractures in the deep tight sandstone reservoir are important for both gas exploration and exploitation activities in Kuqa Depression. We analyzed the characteristics of regional structural evolution and paleotectonic stress setting based on acoustic emission tests and structural feature analysis. Several suites of geomechanical models and experiments were developed to analyze how the geological factors influenced and controlled the development and distribution of fractures during folding. The multilayer model used elasto-plastic finite element method to capture the stress variations and slip along bedding surfaces, and allowed large deformation. The simulated results demonstrate that this novel Quasi-Binary Method coupling composite failure criterion and geomechanical model can effectively quantitatively predict the developed area of fracture parameters in fault-related folds. High-density regions of fractures are mainly located in the fold limbs during initial folding stage, then gradually migrate from forelimb to backlimb, from limbs to hinge, from deep to shallow along with the fold uplift. Among these factors, the fold uplift and slip displacement along fault have the most important influence on distributions of fractures and stress field, meanwhile the lithology and distance to fault have also has certain influences. When the uplift height exceeds approximately 55 percent of the total height of fold the facture density reaches a peak, which conforms to typical top-graben fold type with large amplitude and high-density factures in the top. The overall simulated results match well with core observation and FMI results both in the whole geometry and fracture distribution.