Protein Structure Formation Research Articles

Macromolecular Crowding Enhances Matrix Protein Deposition in Tissue-Engineered Vascular Grafts.

Successful in vitro culture of small-diameter tissue-engineered vascular grafts (TEVGs) requires rapid deposition of biomacromolecules secreted by vascular smooth muscle cells in a polyglycolic acid mesh scaffold's three-dimensional (3D) porous environment. However, common media have lower crowding conditions than in vivo tissue fluids. In addition, during the early stages of construction, most of the biomolecules secreted by the cells into the medium are lost, which negatively affects the TEVG culture process. In this study, we propose the use of macromolecular crowding (MMC) to enhance medium crowding to improve the deposition and self-assembly efficiency of major biomolecules in the early stages of TEVG culture. The addition of carrageenan significantly increased the degree of MMC in the culture medium without affecting cell viability, proliferation, and metabolic activity. Protein analysis demonstrated that the deposition of collagen types I and III and fibronectin increased significantly in the cell layers of two-dimensional and 3D smooth muscle cell cultures after the addition of a MMC agent. Collagen type I in the culture medium decreased significantly compared with that in the medium without a MMC agent. Scanning electron microscopy demonstrated that MMC agents considerably enhanced the formation of matrix protein structures during the early stages of 3D culture. Hence, MMC modifies the crowding degree of the culture medium, resulting in the rapid formation of numerous matrix proteins and fiber structures. Impact Statement Small-diameter tissue-engineered vascular grafts (TEVGs) are one of the most promising means of treating cardiovascular diseases; however, the in vitro construction of TEVGs has some limitations, such as slow deposition of extracellular matrix (ECM), long culture period, and poor mechanical properties. We hypothesized that macromolecular crowding can increase the crowding of the culture medium to construct a more bionic microenvironment, which enhances ECM deposition in the medium to the cell layer and reduces collagen loss, accelerating and enhancing TEVG culture and construction in vitro.

Class A capsid assembly modulator apoptotic elimination of hepatocytes with high HBV core antigen level in vivo is dependent on de novo core protein translation.

Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.

Influence of elevated temperature and high pressure treatments on structural changes of soy protein concentrate

This study focused on investigating the influence of elevated temperature (95–140 °C) and high pressure treatments (30–6000 bar) in different model process systems on protein-protein interactions, particularly in the domain of high-moisture extrusion (HME) conditions, with a specific emphasis on plant-based proteins. The effects of thermomechanical treatments on soy protein concentrate (SPC) were examined at different scales. Macroscopic analysis showed structural changes only at heat treatments exceeding 95 °C, while the application of high pressure alongside elevated temperature treatments did not result in additional changes. On a microscopic scale, protein melting occurred at temperatures above 95 °C, leading to alterations in tertiary and secondary protein structures. For significant protein denaturation and structure formation, a material-temperature threshold of 120 °C for 60% moistened SPC was required. This study sheds light on the key parameters influencing texturization complexity during elevated temperature and high pressure processes like HME. The findings enhance our understanding of protein texturization mechanisms under these conditions.

The Formation of β-Strand Nine (β9) in the Folding and Insertion of BamA from an Unfolded Form into Lipid Bilayers.

Transmembrane proteins span lipid bilayer membranes and serve essential functions in all living cells. Membrane-inserted domains are of either α-helical or β-barrel structure. Despite their biological importance, the biophysical mechanisms of the folding and insertion of proteins into membranes are not well understood. While the relative composition of the secondary structure has been examined by circular dichroism spectroscopy in folding studies for several outer membrane proteins, it is currently not known how individual β-strands fold. Here, the folding and insertion of the β-barrel assembly machinery protein A (BamA) from the outer membrane of Escherichia coli into lipid bilayers were investigated, and the formation of strand nine (β9) of BamA was examined. Eight single-cysteine mutants of BamA were overexpressed and isolated in unfolded form in 8 M urea. In each of these mutants, one of the residues of strand β9, from R572 to V579, was replaced by a cysteine and labeled with the fluorophore IAEDANS for site-directed fluorescence spectroscopy. Upon urea-dilution, the mutants folded into the native structure and were inserted into lipid bilayers of dilauroylphosphatidylcholine, similar to wild-type BamA. An aqueous and a membrane-adsorbed folding intermediate of BamA could be identified by strong shifts in the intensity maxima of the IAEDANS fluorescence of the labeled mutants of BamA towards shorter wavelengths, even in the absence of lipid bilayers. The shifts were greatest for membrane-adsorbed mutants and smaller for the inserted, folded mutants or the aqueous intermediates. The spectra of the mutants V573C-, L575C-, G577C-, and V579C-BamA, facing the lipid bilayer, displayed stronger shifts than the spectra recorded for the mutants R572C-, N574C-, T576C-, and K578C-BamA, facing the β-barrel lumen, in both the membrane-adsorbed form and the folded, inserted form. This alternating pattern was neither observed for the IAEDANS spectra of the unfolded forms nor for the water-collapsed forms, indicating that strand β9 forms in a membrane-adsorbed folding intermediate of BamA. The combination of cysteine scanning mutagenesis and site-directed fluorescence labeling is shown to be a valuable tool in examining the local secondary structure formation of transmembrane proteins.

Ultrasound treatments of tef [Eragrostis tef (Zucc.) Trotter] flour rupture starch α-(1,4) bonds and fragment amylose with modification of gelatinization properties

Tef is a nutritionally-rich ancient grain gaining increasing interest in gluten-free market. Molecular and structural properties in the main biopolymers of two Spanish ecotypes of tef flour physically modified by ultrasound (US) treatments under different temperatures (20, 40, 45, 50 and 55 °C) were studied. Modifications achieved were dependent on tef ecotype, where white tef presented higher susceptibility. Size-exclusion chromatography indicated higher proportion of intermediate amylose chains (degree of polymerization 300–1600) across the whole amylose chain-length distribution and a higher amylose/amylopectin ratio, that increased up to 23% and 19% in treated samples of white and brown tef respectively. US increased the starch short-range order crystallinity and promoted the formation of random coil protein structure and the disappearance of β-sheet, as confirmed from FTIR spectroscopy. XRD and 1H NMR revealed a higher fragmentation of α-(1,4) than α-(1,6) bonds due ultrasonication. The gelatinization temperature range and ΔHgel decreased after ultrasonication up to 6 °C and 13%, an effect that was reinforced or counteracted, respectively, by the treatment temperature, indicating that annealing modulates the impact of sonication on flour gelatinization properties. Tef starch structural properties were found to be significantly modified by US treatments, where temperature was a determining variable influencing the degree of modification achieved.

Combination of pulsed electric field and pH shifting improves the solubility, emulsifying, foaming of commercial soy protein isolate

In this study, the effects of a new dual modification method, pulsed electric field (PEF) and pH shifting treatment, on the solubility, emulsifying and foaming properties of commercial soy protein isolate (SPI) were investigated. Results showed that the combination of PEF and pH shifting treatment, particularly at moderate PEF strength (10 kV/cm) and alkaline conditions (pH 11), facilitated protein structure unfolding and formation of smaller aggregates, substantially enhancing the solubility of SPI from 26.06% to 70.34%. Higher flexibility, surface hydrophobicity, and free sulfhydryl content were also found in the modified SPI, which boosted interfacial activity and significantly enhanced emulsifying and foaming properties. In alkaline environments, the enhanced functional properties of SPI at moderate PEF strength may be attributed to the appropriate oxidation of proteins by free radicals produced by electrochemical processes and the additional unfolding of protein conformations by polarization. The functional characteristics of SPI were decreased by an increase in PEF strength (20 kV/cm), which may be due to the tendency of the protein to aggregate and undergo excessive oxidation. This work may provide an effective methodological and theoretical basis for the modification of SPI by combining PEF and pH shifting treatment. • PEF combined with pH shifting treatment efficiently improved the solubility of SPI. • The treated SPI showed dramatic enhancement in emulsifying and foaming properties. • Enhanced unfolding in pH shifted SPI were observed after PEF introduction. • PEF-induced polarization and proper oxidation enhanced the functional property of SPI.

Impact of microwave radiation on in vitro starch digestibility, structural and thermal properties of rice flour. From dry to wet treatments

Microwave radiation (MW) is an environment-friendly technology used to physically modify flours. Rice flour was MW-treated at different moisture content (MC) (3 %, 8 %, 13 %, 15 %, 20 % and 30 %). In vitro starch digestibility was determined and related to the changes caused by MW treatment to flours' structure and thermal properties, which were influenced by MC. A reduction of 49 % and 65 % in the gelatinization enthalpy of samples treated at 20 % and 30 % MC denoted a partial gelatinization. A loss of granular crystallinity in treated samples was confirmed by XR-diffraction and FTIR, particularly at 15 %, 20 % and 30 % MC. MW promoted the formation of random-coil, α-helix and β-turn protein structure, and the disappearance of LF-β-sheet. Morphological differences were found between samples treated at 8 % MC (loss of polygonal structure, protein layer covering granules' surface and small holes) and 30 % MC (rounded and aggregated granules, covered with exudate amylose). In vitro starch digestibility revealed that samples treated at 20 % and 30 % MC showed 40 % and 47 % higher rapidly digestible starch, 48 % and 70 % lower slowly digestible starch and 90 % lower resistant starch than the untreated flour. Flour MC in MW-treatment allowed the modulation of structural and thermal characteristics of rice flour and consequently its starch hydrolysis rate.

Some useful ideas for multistate protein design: Effect of amino acid substitutions on the multistate proteins stability and the rate of protein structure formation.

The design of new protein variants is usually confined to slightly “fixing” an already existing protein, adapting it to certain conditions or to a new substrate. This is relatively easy to do if the fragment of the protein to be affected, such as the active site of the protein, is known. But what if you need to “fix” the stability of a protein or the rate of its native or intermediate state formation? Having studied a large number of protein mutant forms, we have established the effect of various amino acid substitutions on the energy landscape of the protein. As a result, we have revealed a number of patterns to help researchers identify amino acid residues that determine the folding rate and the stability of globular proteins states and design a mutant form of a protein with desired properties.

Revisiting the concept of peptide bond planarity in an iron-sulfur protein by neutron structure analysis.

The planarity of the peptide bond is important for the stability and structure formation of proteins. However, substantial distortion of peptide bonds has been reported in several high-resolution structures and computational analyses. To investigate the peptide bond planarity, including hydrogen atoms, we report a 1.2-Å resolution neutron structure of the oxidized form of high-potential iron-sulfur protein. This high-resolution neutron structure shows that the nucleus positions of the amide protons deviate from the peptide plane and shift toward the acceptors. The planarity of the H─N─C═O plane depends strongly on the pyramidalization of the nitrogen atom. Moreover, the orientation of the amide proton of Cys75 is different in the reduced and oxidized states, possibly because of the electron storage capacity of the iron-sulfur cluster.

Evaluation of ultrasound-assisted L-histidine marination on beef M. semitendinosus: Insight into meat quality and actomyosin properties

This paper aimed to evaluate the effects of ultrasound-assisted L-histidine marination (UMH) on meat quality and actomyosin properties of beef M. semitendinosus. Our results found that UMH treatment effectively avoided excessive liquid withdrawal, and disrupted myofibril integrity by modifying the water distribution and weakening connection of actin-myosin with increased muscle pH. The ultrasound-treated sample provided more opportunity for the filtration of L-histidine to intervene the isoelectric point and conformation of muscle protein. The activated caspase-3 and changes of ATPase activity in UMH-treated meat accelerated the postmortem ageing, and L-histidine might competitively inhibit the actin-myosin binding by the imidazole group. UMH decreased the surface hydrophobicity by shielding hydrophobic area and unfolding the actomyosin structure. In addition, the increased actomyosin solubility with smaller particle size enhanced the SH content for better cross-linking of myosin tail, and formation of heat-set gelling protein structure. Therefore, UMH treatment manifested the potential to improve beef quality.

Effect of pH and temperature on fibrous structure formation of plant proteins during high-moisture extrusion processing

This study investigated the effect of pH on fibrous structure formation (protein alignment) during high-moisture extrusion processing of gluten, rice protein, as well as pea protein concentrate, and isolate. The pH of the raw material was shifted to 5 and 7 in water suspension with an acid or base and freeze-dried, after which, conductivity, solubility, water-holding capacity, particle size, and pH were measured. The pH-shifted raw materials were extruded at various temperatures (95–160 °C) and the extrudates were analysed for protein alignment (macro and microstructure), tensile strength, free thiol groups, and cooking properties. In general, all raw materials generated fibrous structure at lower temperatures (115–140 °C) at pH 7 than at pH 5 (135–160 °C). Higher pH and temperature values led to an increased tensile strength and pronounced protein alignment. No such unambiguous link could be observed between the raw material properties and enhanced structure formation. This study showed that the structure formation of the extrudate can be positively influenced by increasing the pH of the raw material, which facilitates the plant protein structuring into appealing meat analogue products.

Ultrafast spectroscopy of C-H vibrations in pathogenic bacteria in 3-μm spectral range

Dynamic optical density spectra were obtained under multipulse excitation of bacterial cultures of S. aureus and P. aeruginosa by 3 μm mid-infrared ultrashort laser pulses, corresponding to the vibrational excitation of the C–H bonds of the bacterial cell. These spectra demonstrated pronounced laser intensity-dependent blue spectral shift, presumably associated with the breaking of hydrogen bonds, which are responsible for the formation of secondary and tertiary protein structures.

Effect of pH and heat treatment on structure, surface characteristics and emulsifying properties of purified camel β-casein

Oil-in-water emulsions (20%/80%, w/w) were stabilised by two types of β-caseins (1 g/L, w/w) extracted by rennet coagulation from camel and cow’s milk, respectively. Both extracts were treated under different ranges of pH (3.0, 6.0 and 9.0) and temperature (25, 65 and 95 °C for 15 min) before emulsification. The emulsifying properties of the proteins were studied by surface and interfacial measurements. Results show that the emulsifying activity (EAI) of camel β-casein is higher than the bovine protein. Yet, both proteins exhibited heat stability and nonsignificant effect of temperature was reported. Conversely, a significant effect of pH on camel β-casein was recorded: at pH 6.0, the lowest values of EAI were measured and explained by the formation of micellar protein structure. Under such conditions, camel β-casein is therefore a novel emulsifying protein with high potential to stabilise oil-in-water interfaces which provides numerous applications for the food chemistry field.

Membrane Interactions of α-Synuclein Probed by Neutrons and Photons.

α-Synuclein (α-syn) is a key protein in the etiology of Parkinson's disease. In a disease state, α-syn accumulates as insoluble amyloid fibrils enriched in β-sheet structure. However, in its functional state, α-syn adopts an amphipathic helix upon membrane association and plays a role in synaptic vesicle docking, fusion, and clustering. In this Account, we describe our contributions made in the past decade toward developing a molecular understanding of α-syn membrane interactions, which are crucial for function and have pathological implications. Three topics are covered: α-syn membrane binding probed by neutron reflectometry (NR), the effects of membrane on α-syn amyloid formation, and interactions of α-syn with cellular membranes.NR offers a unique perspective by providing direct measurements of protein penetration depth. By the use of segmentally deuterated α-syn generated through native chemical ligation, the spatial resolution of specific membrane-bound polypeptide regions was determined by NR. Additionally, we used NR to characterize the membrane-bound complex of α-syn and glucocerebrosidase, a lysosomal hydrolase whose mutations are a common genetic risk factor for Parkinson's disease. Although phosphatidylcholine (PC) is the most abundant lipid species in mammalian cells, interactions of PC with α-syn have been largely ignored because they are substantially weaker compared with the electrostatically driven binding of negatively charged lipids. We discovered that α-syn tubulates zwitterionic PC membranes, which is likely related to its involvement in synaptic vesicle fusion by stabilization of membrane curvature. Interestingly, PC lipid tubules inhibit amyloid formation, in contrast to anionic phosphatidylglycerol lipid tubules, which stimulate protein aggregation. We also found that membrane fluidity influences the propensity of α-synuclein amyloid formation. Most recently, we obtained direct evidence of binding of α-syn to exocytic sites on intact cellular membranes using a method called cellular unroofing. This method provides direct access to the cytosolic plasma membrane. Importantly, measurements of fluorescence lifetime distributions revealed that α-syn is more conformationally dynamic at the membrane interface than previously appreciated. This exquisite responsiveness to specific lipid composition and membrane topology is important for both its physiological and pathological functions. Collectively, our work has provided insights into the effects of the chemical nature of phospholipid headgroups on the interplay among membrane remodeling, protein structure, and α-syn amyloid formation.

Effect of Curcumin on Protein Damage Induced by Rotenone in Dopaminergic PC12 Cells.

Oxidative stress is considered to be a key factor of the pathogenesis of Parkinson’s disease, a multifactorial neurodegenerative disorder characterized by reduced dopaminergic neurons in the substantia nigra pars compacta and accumulated protein aggregates. Rotenone is a worldwide-used pesticide that induces the most common features of Parkinson’s by direct inhibition of the mitochondrial complex I. Rotenone-induced Parkinson’s models, as well as brain tissues from Parkinson’s patients, are characterized by the presence of both lipid peroxidation and protein oxidation markers resulting from the increased level of free radical species. Oxidation introduces several modifications in protein structure, including carbonylation and nitrotyrosine formation, which severely compromise cell function. Due to the link existing between oxidative stress and Parkinson’s disease, antioxidant molecules could represent possible therapeutic tools for this disease. In this study, we evaluated the effect of curcumin, a natural compound known for its antioxidant properties, in dopaminergic PC12 cells treated with rotenone, a cell model of Parkinsonism. Our results demonstrate that the treatment of PC12 cells with rotenone causes severe protein damage, with formation of both carbonylated and nitrotyrosine-derived proteins, whereas curcumin (10 µM) co-exposure exerts protective effects by reducing the levels of oxidized proteins. Curcumin also promotes proteasome activation, abolishing the inhibitory effect exerted by rotenone on this degradative system.

Compositional and micromorphological features of hypoplastic enamel in children’s permanent teeth

Enamel hypoplasia is the most common disease of hard tooth tissues of non-carious origin emerging before their eruption. It develops as a result of a delayed and perverse function of ameloblasts leading to the violation of the processes of formation and mineralization of tooth protein structures. The clinical manifestations of enamel hypoplasia are spots, defects in the form of pits, cup-shaped depressions, grooves on various surfaces of child’s teeth. The work is devoted to a comparative investigation of microstructural and compositional features of intact and hypoplastic enamel in children’s permanent teeth using analytical scanning electron microscopy with energy-dispersive X-ray spectroscopy. The hypoplastic enamel is enriched in organic substance and has an irregular structure without keyhole-shaped prismatic structure. Mineral deposition is indicated for hypoplastic enamel including hydroxyapatite with globular structure and calcite and halite, which might have been caused by the violation of extracellular local environment probably due to the enamel hypoplasia development.

An Overview of the Intrinsic Role of Citrullination in Autoimmune Disorders.

A protein undergoes many types of posttranslation modification. Citrullination is one of these modifications, where an arginine amino acid is converted to a citrulline amino acid. This process depends on catalytic enzymes such as peptidylarginine deiminase enzymes (PADs). This modification leads to a charge shift, which affects the protein structure, protein-protein interactions, and hydrogen bond formation, and it may cause protein denaturation. The irreversible citrullination reaction is not limited to a specific protein, cell, or tissue. It can target a wide range of proteins in the cell membrane, cytoplasm, nucleus, and mitochondria. Citrullination is a normal reaction during cell death. Apoptosis is normally accompanied with a clearance process via scavenger cells. A defect in the clearance system either in terms of efficiency or capacity may occur due to massive cell death, which may result in the accumulation and leakage of PAD enzymes and the citrullinated peptide from the necrotized cell which could be recognized by the immune system, where the immunological tolerance will be avoided and the autoimmune disorders will be subsequently triggered. The induction of autoimmune responses, autoantibody production, and cytokines involved in the major autoimmune diseases will be discussed.

Yeast Models for the Study of Amyloid-Associated Disorders and Development of Future Therapy.

First described almost two decades ago, the pioneering yeast models of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases, have become well-established research tools, providing both basic mechanistic insights as well as a platform for the development of therapeutic agents. These maladies are associated with the formation of aggregative amyloid protein structures showing common characteristics, such as the assembly of soluble oligomeric species, binding of indicative dyes, and apoptotic cytotoxicity. The canonical yeast models have recently been expanded by the establishment of a model for type II diabetes, a non-neurological amyloid-associated disease. While these model systems require the exogenous expression of mammalian proteins in yeast, an additional amyloid-associated disease model, comprising solely mutations of endogenous yeast genes, has been recently described. Mutated in the adenine salvage pathway, this yeast model exhibits adenine accumulation, thereby recapitulating adenine inborn error of metabolism disorders. Moreover, in line with the recent extension of the amyloid hypothesis to include metabolite amyloids, in addition to protein-associated ones, the intracellular assembly of adenine amyloid-like structures has been demonstrated using this yeast model. In this review, we describe currently available yeast models of diverse amyloid-associated disorders, as well as their impact on our understanding of disease mechanisms and contribution to future potential drug development.

Rapid Light-Triggered Spatial Reorganization of Proteins in Living Bacteria Cells

In gram-negative bacteria, the cytoplasm is surrounded by two lipid bilayers that encapsulate the peptidoglycan cell wall. Due to a lack of membrane-enclosed organelles within the cytoplasm, the proper formation and spatiotemporal regulation of subcellular protein structures and scaffolds are critical to the cell's ability survive and function. Here we demonstrate, for the first time, that the CRY2/CIB1 system from Arabidopsis thaliana can be used to induce the fast reorganization of proteins inside living bacterialcells. We further show that this system can be used to rapidly inhibit cytokinesis in Escherichia coli cells.

Real-Time Detection of Thiols Using CoPc Modified Black-Phosphorus Based Sensors

Thiols possess a crucial role in the formation of the complex three-dimensional protein structure and are helpful in the inhibition of cellular apoptosis. Enhanced concentration of some thiols can be an indicator of diseases such as cancer, angiogenesis, AIDS, and cardiovascular disease. The chemical details of the living cell and correlation between thiol quantity and associated properties have been of interest to the medical community. It was observed that fluorescein arsenical helix binder –ethanedithiol fluorescence is several times higher than its binding with peptide. Some ethanedithiol derivatives are used to prevent biofilm formation by Proteus Mirabilis and Staphylococcus Epidermidis on medical devices, and hence reducing infections. Similarly, the use of dodecanethiol (DDT) is common in the fabrication of hydrophobic self-assembled monolayers (SAMs), sensors, derivatization applications, and environment monitoring. In this report, two different thiols (ethanedithiol and dodecanethiol) were detected using CoPc (cobalt phthalocyanine) -black-P type hybrid sensors. Electrochemical tests of thiols-based buffers using specially designed electrodes such as Si-Ag-Black P-CoPc and AgIC-Ag-Black-P-CoPc were performed. Many of these electrodes exhibited a systematic response when thiol concentration was varied from ∼ 1.0 to 5.0 μL/mL. Vapor phase real time sensing of the thiol also exhibited very good performance. The Randles-Sevcik equation, which linearly relates the peak current and the square root of potential scan rate as observed in cyclic voltammetry tests, was used in performance evaluations. Electrochemical impedance spectroscopy results suggest that CoPc coating helps to enhance electron transfer capability of black-P.