Unfolded Chains Research Articles

Delineating, Imaging, and Assessing Pulmonary Fibrosis Remodeling via Collagen Hybridization.

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening disease with no early detection, few treatments, and dismal outcomes. Although collagen overdeposition is a hallmark of lung fibrosis, current research mostly focuses on the cellular aspect, leaving collagen, particularly its dynamic remodeling (i.e., degradation and turnover), largely unexplored. Here, using a collagen hybridizing peptide (CHP) that specifically binds unfolded collagen chains, we reveal vast collagen denaturation in human IPF lungs and delineate the spatiotemporal progression of collagen denaturation three-dimensionally within fibrotic lungs in mice. Transcriptomic analyses support that lung collagen denaturation is strongly associated with up-regulated collagen catabolism in mice and patients. We thus show that CHP probing differentiates remodeling responses to antifibrotics and highlights the resolution of established fibrosis by agents up-regulating collagen catabolism. We further develop a radioactive CHP that detects fibrosis in vivo in mice as early as 7 days postlung-injury (Ashcroft score: 2-3) by positron emission tomography (PET) imaging and ex vivo in clinical lung specimens. These findings establish collagen denaturation as a promising marker of fibrotic remodeling for the investigation, diagnosis, and therapeutic development of pulmonary fibrosis.

Pathogenesis of human atheroma necrotic core: degradation of connective tissue fibers and possible involvement of cathepsin K

BackgroundAtheroma is a serious atherosclerotic lesion related to plaque rupture, thrombosis, and ischemic disease. To clarify the pathogenesis of human atheroma, particularly the formation of the liponecrotic tissue in the necrotic core, the distribution of connective tissue fibers, such as collagen and elastic fibers, and related substances was investigated in this study.MethodsAtherosclerotic lesions in human coronary arteries were classified into three categories: pathologic intimal thickening (PIT), atheroma with lipid core (ALC), and atheroma with necrotic core (ANC). PIT and ALC consisted of the lipid pool and the lipid core, respectively. The necrotic core of ANC was composed of a lipid core-like region and liponecrotic tissue containing amorphous materials and lacking cells and connective tissue fibers. The distribution of collagen type I, elastin, C-terminal crosslinked telopeptide of collagen type I (CTX-I), and cathepsin K (CatK) was investigated through immunohistochemistry. CTX-I is a fragmented peptide consisting of the C-terminal region of collagen type I that is generated by CatK. The distribution of denatured and unfolded collagen chains was also investigated through collagen hybridizing peptide staining.ResultsCollagen type I, denatured and unfolded collagen chains, and elastin were positively stained in the PIT, ALC, and lipid core-like region of ANC. However, they were negatively stained in the liponecrotic tissue of ANC. CTX-I and CatK were positively stained in all four regions, and their grade of staining appeared to show a positive relationship. Both CTX-I and CatK demonstrated higher staining grades in the liponecrotic tissue. These findings suggested that pre-existing collagen type I was severely degraded by CatK, resulting in the production of CTX-I in the liponecrotic tissue, and that pre-existing elastin was also degraded in this region. CatK was mainly found in macrophage foam cells, many of which were localized within and around the liponecrotic tissue.ConclusionsThis study suggests that the liponecrotic tissue in the necrotic core of human atheroma is formed by severe degradation of pre-existing connective tissue fibers and consequent collapse of tissue structure. This degradation is likely caused by proteolytic enzymes, such as CatK, secreted by macrophage foam cells.

The ribosome lowers the entropic penalty of protein folding

Most proteins fold during biosynthesis on the ribosome1, and co-translational folding energetics, pathways and outcomes of many proteins have been found to differ considerably from those in refolding studies2–10. The origin of this folding modulation by the ribosome has remained unknown. Here we have determined atomistic structures of the unfolded state of a model protein on and off the ribosome, which reveal that the ribosome structurally expands the unfolded nascent chain and increases its solvation, resulting in its entropic destabilization relative to the peptide chain in isolation. Quantitative 19F NMR experiments confirm that this destabilization reduces the entropic penalty of folding by up to 30 kcal mol−1 and promotes formation of partially folded intermediates on the ribosome, an observation that extends to other protein domains and is obligate for some proteins to acquire their active conformation. The thermodynamic effects also contribute to the ribosome protecting the nascent chain from mutation-induced unfolding, which suggests a crucial role of the ribosome in supporting protein evolution. By correlating nascent chain structure and dynamics to their folding energetics and post-translational outcomes, our findings establish the physical basis of the distinct thermodynamics of co-translational protein folding.

Stabilization of Non-Native Folds and Programmable Protein Gelation in Compositionally Designed Deep Eutectic Solvents.

Proteins are adjustable units from which biomaterials with designed properties can be developed. However, non-native folded states with controlled topologies are hardly accessible in aqueous environments, limiting their prospects as building blocks. Here, we demonstrate the ability of a series of anhydrous deep eutectic solvents (DESs) to precisely control the conformational landscape of proteins. We reveal that systematic variations in the chemical composition of binary and ternary DESs dictate the stabilization of a wide range of conformations, that is, compact globular folds, intermediate folding states, or unfolded chains, as well as controlling their collective behavior. Besides, different conformational states can be visited by simply adjusting the composition of ternary DESs, allowing for the refolding of unfolded states and vice versa. Notably, we show that these intermediates can trigger the formation of supramolecular gels, also known as eutectogels, where their mechanical properties correlate to the folding state of the protein. Given the inherent vulnerability of proteins outside the native fold in aqueous environments, our findings highlight DESs as tailorable solvents capable of stabilizing various non-native conformations on demand through solvent design.

Proline Peptide Bond Isomerization in Ubiquitin Under Folding and Denaturing Conditions by Pressure-Jump NMR

Proline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kcal/mole for denatured polypeptides but showed a substantial drop to 12.7 kcal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized ‘folding nucleus’. For cis-P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensity revealed a slow component attributed to cis → trans isomerization of P37. This result implies that the NMR-invisible cis-P37 isomer adopts a molten globule state that encompasses the entire length of the ubiquitin chain, suggestive of a structure that mostly resembles the folded state.

Transient physics in the compression and mixing dynamics of two nanochannel-confined polymer chains.

We use molecular dynamics (MD) simulation and nanofluidic experiments to probe the non-equilibrium transient physics of two nanochannel-confined polymers driven against a permeable barrier in a flow field. For chains with a persistence length P smaller than the channel diameter D, both simulation and experiment with dsDNA reveal nonuniform mixing of the two chains, with one chain dominating locally in what we term "aggregates." Aggregates undergo stochastic dynamics, persisting for a limited time, then disappearing and reforming. Whereas aggregate-prone mixing occurs immediately at sufficiently high flow speeds, chains stay segregated at intermediate flow for some time, often attempting to mix multiple times, before suddenly successfully mixing. Observation of successful mixing nucleation events in nanofluidic experiments reveal that they arise through a peculiar "back-propagation" mechanism whereby the upstream chain, closest to the barrier, penetrates and passes through the downstream chain (farthest from the barrier) moving against the flow direction. Simulations suggest that the observed back-propagation nucleation mechanism is favored at intermediate flow speeds and arises from a special configuration where the upstream chain exhibits one or more folds facing the downstream chain, while the downstream chain has an unfolded chain end facing upstream.

Effects of altered backbone composition on the folding kinetics and mechanism of an ultrafast-folding protein.

Sequence-encoded protein folding is a ubiquitous biological process that has been successfully engineered in a range of oligomeric molecules with artificial backbone chemical connectivity. A remarkable aspect of protein folding is the contrast between the rapid rates at which most sequences in nature fold and the vast number of conformational states possible in an unfolded chain with hundreds of rotatable bonds. Research efforts spanning several decades have sought to elucidate the fundamental chemical principles that dictate the speed and mechanism of natural protein folding. In contrast, little is known about how protein mimetic entities transition between an unfolded and folded state. Here, we report effects of altered backbone connectivity on the folding kinetics and mechanism of the B domain of Staphylococcal protein A (BdpA), an ultrafast-folding sequence. A combination of experimental biophysical analysis and atomistic molecular dynamics simulations performed on the prototype protein and several heterogeneous-backbone variants reveal the interplay among backbone flexibility, folding rates, and structural details of the transition state ensemble. Collectively, these findings suggest a significant degree of plasticity in the mechanisms that can give rise to ultrafast folding in the BdpA sequence and provide atomic level insights into how protein mimetic chains adopt an ordered folded state.

Event Segmentation in the Audio Description of Films

To make the content of films available to a visually impaired audience, a sighted translator can provide audio description (AD), a verbal description of visual events. To achieve this goal, the audio describer needs to select what to describe, when to describe it, and how to describe it, as well as to express the information aurally. The efficacy of this communication is critically dependent upon basic cognitive processes of how the sighted audio describer perceives and segments the film’s unfolding chain of events and in what way the visually impaired end users conceive the structure, content, and segmentation of such events in relation to the produced AD. There is, however, virtually no research on this interplay in relation to AD. In this study, we scrutinize live AD of a film from two trained audio describers and examine how events are structured, segmented and construed in their AD. Results demonstrate that the event segmentation experienced from the film is indeed a fundamental part of how AD is structured and construed. It was found that AD at event boundaries was highly sensitive to different spatiotemporal circumstances and this relationship depends on semantic resources for expressing AD. Lay summary In our everyday lives, we naturally organize and remember our experiences as meaningful sequences of events. This happens the same way when we watch movies – we see them as a series of events happening in different places, times, and with changing character dynamics. Now, think about making this cinematic experience accessible to people with visual impairments. This is where a sighted translator, called an audio describer, becomes really important. To make movies understandable for a visually impaired audience, a sighted audio describer describes crucial visual events in the film. Their goal is to create a "narrative equivalence" between the original film and the audio-described version. Thus, the main challenges for audio describers are deciding what information to describe, when to do it, and how to articulate it effectively. In the current case study, we looked at two audio descriptions for the Swedish film "Skumtimmen" (English title: "Echoes from the Dead"). These descriptions were created by trained audio describers. We wanted to see how these narrators organized, broke down, and verbalized the course of events in their audio descriptions. To achieve this, we focused on two important parts of storytelling – changes in time and location – and paid special attention to how often and in what way audio describers verbalized these event boundaries. What we found is that both describers indeed verbalized most event boundaries. This shows that these boundaries are a big part of how audio descriptions are put together. We also learned that the descriptions at these event boundaries were very sensitive to when and where key events unfolded in the story. The way these boundaries were described depended on fundamental cognitive and linguistic resources to talk about space and time. Our findings help us understand how breaking down events in a film is a crucial part of shaping audio descriptions. Plus, our research highlights how important it is to study how events are broken down and verbalized, giving useful insights to improve audio descriptions and train future audio describers. The ultimate goal is to make the experience better for visually impaired viewers, contributing to a more inclusive and enjoyable audiovisual world.

Integrated trophic position as a proxy for food‐web complexity

Abstract There are two distinct approaches to describing the distributions of biomass and species in food webs: one to consider them as discrete trophic levels (TLs); and the other to consider them as continuous trophic positions (TPs). Bridging the gap between these two perspectives presents a nontrivial challenge in integrating biodiversity and food‐web structure. Food network unfolding (FNU) is a technique used to bridge this gap by partitioning the biomass of species into integer TLs to compute three complexity indices, namely vertical (DV), horizontal (DH) and range (DR) diversity (D indices), through decomposition of Shannon's index H′. Using FNU, the food web (a network of species with unique TPs) is converted to a linear food chain (a biomass distribution at discrete TLs). This enables us to expect that the unfolded biomass within species decreases exponentially as the TL increases. Under this condition, the mean TL value in unfolded food chains is hypothesized to have an exponential relationship with the vertical diversity, DV. To explore this, we implemented FNU and calculated D indices for food webs publicly available at EcoBase (n = 158) and calculated the integrated TP (iTP), defined as the biomass‐weighted average TP of a given food web. The iTP corresponds to the mean TL in unfolded food chains and can be empirically measured through compound‐specific isotope analysis of amino acids (CSIA‐AA). Although our analysis is biased towards marine ecosystems, we revealed an exponential relationship between iTP and DV, suggesting that iTP can serve as a measurable proxy for DV. Furthermore, we found a positive correlation between the iTP observed in the total communities (total iTP) and the iTPs of partial communities consisting only of species with 2.0 ≤ TP < 3.0 (partial iTP; r2 = 0.48), suggesting that DV can be predicted using partial iTP. Our findings suggest that the net effect of species diversity, excluding the effect of biomass (corresponding to H′ − DV), on food‐web complexity can be revealed by combining CSIA‐AA with biodiversity analysis (e.g. environmental DNA).

Local and Large-Scale Conformational Dynamics in Unfolded Proteins and IDPs. II. Effect of Temperature and Internal Friction.

Internal dynamics of proteins are essential for protein folding and function. Dynamics in unfolded proteins are of particular interest since they are the basis for many cellular processes like folding, misfolding, aggregation, and amyloid formation and also determine the properties of intrinsically disordered proteins (IDPs). It is still an open question of what governs motions in unfolded proteins and whether they encounter major energy barriers. Here we use triplet-triplet energy transfer (TTET) in unfolded homopolypeptide chains and IDPs to characterize the barriers for local and long-range loop formation. The results show that the formation of short loops encounters major energy barriers with activation energies (Ea) up to 18 kJ/mol (corrected for effects of temperature on water viscosity) with very little dependence on amino acid sequence. For poly(Gly-Ser) and polySer chains the barrier decreases with increasing loop size and reaches a limiting value of 4.6 ± 0.4 kJ/mol for long and flexible chains. This observation is in accordance with the concept of internal friction encountered by chain motions due to steric effects, which is high for local motions and decreases with increasing loop size. Comparison with the results from the viscosity dependence of loop formation shows a negative correlation between Ea and the sensitivity of the reaction to solvent viscosity (α) in accordance with the Grote-Hynes theory of memory friction. The Arrhenius pre-exponential factor (A) also decreases with increasing loop size, indicating increased entropic costs for loop formation. Long-range loop formation in the investigated sequences derived from IDPs shows increased Ea and A compared with poly(Gly-Ser) and polySer chains. This increase is exclusively due to steric effects that cause additional internal friction, whereas intramolecular hydrogen bonds, dispersion forces, and charge interactions do not affect the activation parameters.

Cooperative & competitive binding of anti-myosin tail antibodies revealed by super-resolution microscopy

The MF30 monoclonal antibody, which binds to the myosin subfragment-2 (S2), was found to increase the extent of myofibril shortening. Yet, previous observations found no effect of this antibody on actin sliding over myosin during in vitro motility assays with purified proteins in which myosin binding protein C (MyBPC) was absent. MF30 is hypothesized to enhance the availability of myosin heads (subfragment-1 or S1) to bind actin by destabilizing the myosin S2 coiled-coil and sterically blocking S2 from binding S1. The mechanism of action likely includes MF30's substantial size, thereby inhibiting S1 heads and MyBPC from binding S2. Hypothetically, MF30 should enhance the ON state of myosin, thereby increasing muscle contraction. Our findings indicate that MF30 binds preferentially to the unfolded heavy chains of S2, displaying positive cooperativity. However, the dose-response curve of MF30's enhancement of myofibril shortening did not suggest complex interactions with S2. Single, double, and triple-stained myofibrils with increasing amounts of antibodies against myosin rods indicate a possible competition with MyBPC. Additional assays revealed decreased fluorescence intensity at the C-zone (central zone in the sarcomere, where MyBPC is located), where MyBPC may inhibit MF30 binding. Another monoclonal antibody named MF20, which binds to the light meromyosin (LMM) without affecting myofibril contraction, showed less reduction in fluorescence intensity at the C-zone in expansion microscopy than MF30. Expansion microscopy images of myofibrils labeled with MF20 revealed labeling of the A-band (anisotropic band) and a slight reduction in the labeling at the C-zone. The staining pattern obtained from the expansion microscopy image was consistent with images from photolocalization microscopy which required the synthesis of unique photoactivatable quantum dots, and Zeiss Airyscan imaging as well as alternative expansion microscopy digestion methods. Consistent with the hypothesis that MF30 competes with MyBPC binding to S2, cardiac tissue from MyBPC knockout mice was stained more intensely, especially in the C-zone, by MF30 compared to the wild type.

Paradoxical Effects of Endoplasmic Reticulum Aminopeptidase 1 Deficiency on HLA-B27 and Its Role as an Epistatic Modifier in Experimental Spondyloarthritis.

We undertook this study to examine the functional basis for epistasis between endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 in experimental spondyloarthritis (SpA). ERAP1-knockout rats were created using genome editing and bred with HLA-B27/human β2 -microglobulin-transgenic (HLA-B27-Tg) rats and HLA-B7-Tg rats. The effects of ERAP1 deficiency on HLA allotypes were determined using immunoprecipitation and immunoblotting, flow cytometry, allogeneic T cell proliferation assays, and gene expression analyses. Animals were examined for clinical features of disease, and tissue was assessed by histology. ERAP1 deficiency increased the ratio of folded to unfolded (β2 m-free) HLA-B27 heavy chains, while having the opposite effect on HLA-B7. Furthermore, in rats with ERAP1 deficiency, HLA-B27 misfolding was reduced, while free HLA-B27 heavy chain dimers on the cell surface and monomers were increased. The effects of ERAP1 deficiency persisted during up-regulation of HLA-B27 and led to a reduction in endoplasmic reticulum stress. ERAP1 deficiency reduced the prevalence of arthritis in HLA-B27-Tg rats by two-thirds without reducing gastrointestinal inflammation. Dendritic cell abnormalities attributed to the presence of HLA-B27, including reduced allogeneic T cell stimulation and loss of CD103-positive/major histocompatibility complex class II-positive cells, were not rescued by ERAP1 deficiency, while excess Il23a up-regulation was mitigated. ERAP1 deficiency reduced HLA-B27 misfolding and improved folding while having opposing effects on HLA-B7. The finding that HLA-B27-Tg rats had partial protection against SpA in this study is consistent with genetic evidence that loss-of-function and/or reduced expression of ERAP1 reduces the risk of ankylosing spondylitis. Functional studies support the concept that the effects of ERAP1 on HLA-B27 and SpA may be a consequence of how peptides affect the biology of this allotype rather than their role as antigenic determinants.

Direct observation of Hsp90-induced compaction in a protein chain.

The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using optical tweezers, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded, and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable clients and cooperates with Hsp70.

High-Moisture Shear Processes: Molecular Changes of Wheat Gluten and Potential Plant-Based Proteins for Its Replacement.

Nowadays, a growing offering of plant-based meat alternatives is available in the food market. Technologically, these products are produced through high-moisture shear technology. Process settings and material composition have a significant impact on the physicochemical characteristics of the final products. Throughout the process, the unfolded protein chains may be reduced, or associate in larger structures, creating rearrangement and cross-linking during the cooling stage. Generally, soy and pea proteins are the most used ingredients in plant-based meat analogues. Nevertheless, these proteins have shown poorer results with respect to the typical fibrousness and juiciness found in real meat. To address this limitation, wheat gluten is often incorporated into the formulations. This literature review highlights the key role of wheat gluten in creating products with higher anisotropy. The generation of new disulfide bonds after the addition of wheat gluten is critical to achieve the sought-after fibrous texture, whereas its incompatibility with the other protein phase present in the system is critical for the structuring process. However, allergenicity problems related to wheat gluten require alternatives, hence an evaluation of underutilized plant-based proteins has been carried out to identify those that potentially can imitate wheat gluten behavior during high-moisture shear processing.

Rosemary Extract-Induced Autophagy and Decrease in Accumulation of Collagen Type I in Osteogenesis Imperfecta Skin Fibroblasts.

Osteogenesis imperfecta (OI) is a heterogeneous connective tissue disease mainly caused by structural mutations in type I collagen. Mutant collagen accumulates intracellularly, causing cellular stress that has recently been shown to be phenotype-related. Therefore, the aim of the study was to search for potential drugs reducing collagen accumulation and improving OI fibroblast homeostasis. We found that rosemary extract (RE), which is of great interest to researchers due to its high therapeutic potential, at concentrations of 50 and 100 µg/mL significantly reduced the level of accumulated collagen in the fibroblasts of four patients with severe and lethal OI. The decrease in collagen accumulation was associated with RE-induced autophagy as was evidenced by an increase in the LC3-II/LC3-I ratio, a decrease in p62, and co-localization of type I collagen with LC3-II and LAMP2A by confocal microscopy. The unfolded protein response, activated in three of the four tested cells, and the level of pro-apoptotic markers (Bax, CHOP and cleaved caspase 3) were attenuated by RE. In addition, the role of RE-modulated proteasome in the degradation of unfolded procollagen chains was investigated. This study provides new insight into the beneficial effects of RE that may have some implications in OI therapy targeting cellular stress.

Modulating co-translational protein folding by rational design and ribosome engineering

Co-translational folding is a fundamental process for the efficient biosynthesis of nascent polypeptides that emerge through the ribosome exit tunnel. To understand how this process is modulated by the shape and surface of the narrow tunnel, we have rationally engineered three exit tunnel protein loops (uL22, uL23 and uL24) of the 70S ribosome by CRISPR/Cas9 gene editing, and studied the co-translational folding of an immunoglobulin-like filamin domain (FLN5). Our thermodynamics measurements employing 19F/15N/methyl-TROSY NMR spectroscopy together with cryo-EM and molecular dynamics simulations reveal how the variations in the lengths of the loops present across species exert their distinct effects on the free energy of FLN5 folding. A concerted interplay of the uL23 and uL24 loops is sufficient to alter co-translational folding energetics, which we highlight by the opposite folding outcomes resulting from their extensions. These subtle modulations occur through a combination of the steric effects relating to the shape of the tunnel, the dynamic interactions between the ribosome surface and the unfolded nascent chain, and its altered exit pathway within the vestibule. These results illustrate the role of the exit tunnel structure in co-translational folding, and provide principles for how to remodel it to elicit a desired folding outcome.

Backbone N-Amination Promotes the Folding of β-Hairpin Peptides via a Network of Hydrogen Bonds.

Molecular dynamics (MD) simulations have been used to characterize the effects of backbone N-amination of residues in a model β-hairpin peptide. This modification is of considerable interest as N-aminated peptides have been shown to inhibit amyloid-type aggregation. Six derivatives of the β-hairpin peptide, which contain one, two, or four N-aminated residues, have been studied. For each peptide 100 ns MD simulations starting from the folded β-hairpin structure were performed. The effects of the N-amination prove to be very sequence dependent. N-Amination of a residue involved in interstrand hydrogen bonding (Val3) leads to unfolding of the β-hairpin, whereas N-amination of a residue toward the C-terminus (Leu11) gives fraying at the termini of the peptide. In the other derivatives the peptide remains folded, with increasing levels of N-amination reducing the right-handed twist of the β-hairpin and favoring population of a type II' rather than a type I' β-turn. MD simulations (100 ns) have also been run for each peptide starting from an unfolded extended chain. Here, the peptide with four N-aminated residues shows the most folding into the β-hairpin (34%). Analysis of the simulations shows that N-amination favors the population of β (φ, ψ) conformations by the preceding residue due to, at least in part, a network of weak NH2(i)-CO(i) and NH2(i)-CO(i-2) hydrogen bonds. It also leads to a reduction of misfolding because of changes in the hydrogen-bonding potential. Both of these features help funnel the peptide to the folded β-hairpin structure. The conformational insights provided through this work give a firm foundation for the design of N-aminated peptide inhibitors for modulating protein-protein interactions and aggregation.

High-efficiency casing perforation oil and gas wells

The energetic capabilities of a high-speed jet of an aqueous solution of polyethylene oxide (PEO) with varying concentration and different outflow pressures from a jetforming nozzle were investigated using the length of the forming channel in the model of the casing of an oil and gas well, cement sheath and rock layer, as well as impact of the jet force on a metal plate fixed on a physical pendulum. The experimental data made it possible to obtain a calculated dependence in a dimensionless form to determine the quality (initial sections) of jets of aqueous solutions with different concentrations and molecular weights of PEO, considering the real parameters of the jet-forming nozzles of the hydroperforator. A comprehensive study of the perforation process made it possible to substantiate the mechanism of the high destructive capacity of a high-speed jet of polymer solution. It has been established that the mechanism of the high destructive capacity of the polymer water jet is not due to the Toms effect, but caused by the destructive action of the dynamic pressure of the polymer water jet «reinforced» by strongly unfolded macromolecular chains under the action of a tensile flow in the inlet area of the jet forming nozzle of the hydroperforator. Keywords: perforator; jet nozzle; jet quality; casing; cement sheath; rock; Toms effect.

Quantitative Description of Intrinsically Disordered Proteins Using Single-Molecule FRET, NMR, and SAXS.

Studying the conformational landscape of intrinsically disordered and partially folded proteins is challenging and only accessible to a few solution state techniques, such as nuclear magnetic resonance (NMR), small-angle scattering techniques, and single-molecule Förster resonance energy transfer (smFRET). While each of the techniques is sensitive to different properties of the disordered chain, such as local structural propensities, overall dimension, or intermediate- and long-range contacts, conformational ensembles describing intrinsically disordered proteins (IDPs) accurately should ideally respect all of these properties. Here we develop an integrated approach using a large set of FRET efficiencies and fluorescence lifetimes, NMR chemical shifts, and paramagnetic relaxation enhancements (PREs), as well as small-angle X-ray scattering (SAXS) to derive quantitative conformational ensembles in agreement with all parameters. Our approach is tested using simulated data (five sets of PREs and 15 FRET efficiencies) and validated experimentally on the example of the disordered domain of measles virus phosphoprotein, providing new insights into the conformational landscape of this viral protein that comprises transient structural elements and is more compact than an unfolded chain throughout its length. Rigorous cross-validation using FRET efficiencies, fluorescence lifetimes, and SAXS demonstrates the predictive nature of the calculated conformational ensembles and underlines the potential of this strategy in integrative dynamic structural biology.

Interactions between nascent proteins and the ribosome surface inhibit co-translational folding

Most proteins begin to fold during biosynthesis on the ribosome. It has been suggested that interactions between the emerging polypeptide and the ribosome surface might allow the ribosome itself to modulate co-translational folding. Here we combine protein engineering and NMR spectroscopy to characterize a series of interactions between the ribosome surface and unfolded nascent chains of the immunoglobulin-like FLN5 filamin domain. The strongest interactions are found for a C-terminal segment that is essential for folding, and we demonstrate quantitative agreement between the strength of this interaction and the energetics of the co-translational folding process itself. Mutations in this region that reduce the extent of binding result in a shift in the co-translational folding equilibrium towards the native state. Our results therefore demonstrate that a competition between folding and binding provides a simple, dynamic mechanism for the modulation of co-translational folding by the ribosome.