Designing collagens to shed light on the multi-scale structure-function mapping of matrix disorders.

Collagens are the most abundant structural proteins in the extracellular matrix of animals and play crucial roles in maintaining the structural integrity and mechanical properties of tissues and organs while mediating important biological processes. Fibrillar collagens have a unique triple helix structure with a characteristic repeating sequence of (Gly-X-Y)n. Variations within the repetitive sequence can cause misfolding of the triple helix, resulting in heritable connective tissue disorders. The most common variations are single-point missense mutations that lead to the substitution of a glycine residue with a bulkier amino acid (Gly â†’ X). In this review, we will first discuss the importance of collagen's triple helix structure and how single Gly substitutions can impact its folding, structure, secretion, assembly into higher-order structures, and biological functions. We will review the role of "designer collagens," i.e., synthetic collagen-mimetic peptides and recombinant bacterial collagen as model systems to include Gly â†’ X substitutions observed in collagen disorders and investigate their impact on structure and function utilizing in vitro studies. Lastly, we will explore how computational modeling of collagen peptides, especially molecular and steered molecular dynamics, has been instrumental in probing the effects of Gly substitutions on structure, receptor binding, and mechanical stability across multiple length scales.

Image analysis-based discoloration rate quantification and kinetic modeling for shelf-life prediction in herb-coated pear slices.

The present research study aimed to examine three different herb extract's effects on the discoloration rate of fresh-cut pear slices using an image analysis technique. Pear slices were sprayed and dip-coated with Ocimum basilicum, Origanum vulgare, and Camellia sinensis (0.1 g/ml) extract solution. During 15 days storage period with three days intervals, all sprayed/dip-coated pear slices were analyzed for the quality attribute (TA) and color parameters notably a*, b*, hue angle (H*), lightness (L*), and total color change (ΔE). Further, order kinetic models were used to observe the color changes and to predict the shelf-life. The results obtained showed that the applicability of image analysis helped to predict the discoloration rate, and it was better fitted to the first-order (FO) kinetic model (R2 ranging from 0.87 to 0.99). Based on the kinetic model, color features ΔE and L* was used to predict the shelf-life as they had high regression coefficient values. Thus, the findings obtained from the kinetic study demonstrated Camellia sinensis (assamica) extract spray-coated pear slices reported approximately 28.63- and 27.95-days shelf-stability without much discoloration compared with all other types of surface coating.

The energetics and evolution of oxidoreductases in deep time.

The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation-reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox (https://protein-redox-potential.web.app), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation-reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user-contributed submissions with the intention of making it a valuable resource for researchers in this field.

Comparative studies on the rheological characteristics, functional attributes, and baking stability of xanthan and guar gum formulated honey gel matrix.

The research aims to enhance the characteristics of honey by incorporating xanthan gum (XG) and guar gum (GG) at various concentrations (0.5-2.0% w/w) and preparing a honey gel matrix (HGM) through high-shear homogenization. This approach serves as a substitute for fat-based filling materials commonly used in bakery products. The study encompassed an investigation of the rheological characteristics (steady and dynamic), total phenolic content (TPC), antioxidant activity, and baking stability of the HGMs. The concentration of the gums used significantly influenced the transformation of honey into the HGM and its stability. Notably, the XG-HGM demonstrated greater shear thinning behavior and higher consistency compared to the GG-HGM. Herschel Bulkley and power law models were found to be the best-fitted models for XG-HGM and GG-HGM, respectively. Furthermore, both XG-HGM and GG-HGM exhibited a higher viscous component (G″) than an elastic component (G') at low concentrations, up to 1% (w/w) for XG-HGM and 1.5% (w/w) for GG-HGM; however, this behavior reversed beyond those concentrations (G' > G″). The XG-HGM exhibited lower temperature sensitivity compared to GG-HGM, indicating better stability under varying heat conditions. Moreover, both TPC and antioxidant activity decreased with increasing concentrations of both gums. The XG-HGM achieved the highest baking stability index, reaching 95.23% at a 2% concentration. This modified HGM formulated with XG demonstrated superior consistency, color retention, and exceptional baking stability, making it a promising candidate for application as a filling material in the bakery sector. Its improved stability and quality can facilitate the development of a wide range of baking products in the food industry.

Comprehensive Analysis of Physicochemical, Functional, Thermal, and Morphological Properties of Microgreens from Different Botanical Sources.

Due to the significant increase in global pollution and a corresponding decrease in agricultural land, there is a growing demand for sustainable modes of modern agriculture that can provide nutritious food. In this regard, microgreens are an excellent option as they are loaded with nutrients and can be grown in controlled environments using various vertical farming approaches. Microgreens are salad crops that mature within 15-20 days, and they have tender leaves with an abundant nutritive value. Therefore, this study aims to explore the physicochemical, techno-functional, functional, thermal, and morphological characteristics of four botanical varieties of microgreens, including carrot (Daucus carota), spinach (Spinacia oleracea), bathua (Chenopodium album), and Bengal gram (Cicer arietinum), which are known for their exceptional nutritional benefits. Among the four botanical varieties of microgreens studied, bathua microgreens demonstrated the highest protein content (3.40%), water holding capacity (1.58 g/g), emulsion activity (56.37%), and emulsion stability (53.72%). On the other hand, Bengal gram microgreens had the highest total phenolic content (32.2 mg GAE/g), total flavonoid content (7.57 mg QE/100 g), and DPPH activity (90.60%). Fourier transform infrared spectroscopy analysis of all microgreens revealed the presence of alkanes, amines, and alcohols. Moreover, X-ray diffraction analysis indicated low crystallinity and high amorphousness in the microgreens. Particle size analysis showed that the median, modal, and mean sizes of the microgreens ranged from 110.327 to 952.393, 331.06 to 857.773, and 97.567 to 406.037 µm, respectively. As per the observations of the results, specific types of microgreens can be utilized as an ingredient in food processing industry, including bakery, confectionery, and more, making them a promising nutritive additive for consumers. This study sheds light on various food-based analytical parameters and offers a foundation for future research to fully harness the potential of microgreens as a novel and sustainable food source, benefiting both the industry and consumers alike.

Anti-cancerous effect of corn silk: a critical review on its mechanism of action and safety evaluation.

Cancer is a broad collection of diseases that can begin in almost any organ or tissue of the body. Corn silk is the hair-like stigmata of female maize flowers which is generally discarded as waste from maize cultivation. The current study targets the anti-cancer potential of corn silk and its bioactive compounds namely, polyphenols, flavonoids, and sterols. The polyphenols and flavonoids like quercetin, rutin, apigenin and beta-sitosterol are a range of compounds from corn silk which were investigated for their anticancer effect. Corn silk showed apoptotic and antiproliferative effects in cancer cells through different signalling pathways, essentially the serine/threonine kinases (Akt)/lipid kinases (PI3Ks) pathway. The study revealed that corn silk compounds target immune cell responses, induce cell cytotoxicity, and upregulate the expression of proapoptotic genes p53, p21, caspase 9, and caspase 3 in certain cancer cell lines including HeLa cervical cancer cells, MCF-7 breast cancer cells, PANC-02 pancreatic cancer cells and Caco-2 colon cancer cells. Flavonoids derived from corn silk enhance T cell mediated immune response and decrease inflammatory factors. Corn silk bioactive compounds were found to reduce the side effects of cancer therapy. Antioxidants of corn silk, quercetin and rutin help in reducing the nephrotoxicity of chemotherapeutic drugs. The study also suggests that corn silk has anti-cancerous potential as it targets tumour suppression and inhibits metastasis A dose of 500 mg/kg body weight of corn silk has been found safe for human consumption. Corn silk extract can be used as a preventive or therapeutic step to cure cancer. The anti-cancer property, mechanism and role of corn silk in controlling cancer-related side effects have been critically reviewed providing new scope for the use of corn silk in cancer therapy.

Bioactive compounds of corn silk and their role in management of glycaemic response.

Management of glycaemic response is perhaps the most critical part of antidiabetic therapy. Hypoglycaemia is an avoidable complication caused by conventional drugs used in the treatment of diabetes. It triggers commonly during the intensification of anti-hyperglycemic therapy used to render glycemic control in diabetic patients. The commercial oral hypoglycaemic drugs, insulin, herbal medicines and plant extracts are therefore used as a part of the treatment of diabetes. The demand for treating diabetes, through herbal and plant resources is due to their lesser adverse reactions and better phytochemical benefits. Corn silk has been shown to have anti-allergic, anti-inflammatory, and anti-hypertensive effects when extracted in various solvents. Corn silk has medicinal characteristics and has long been used as a traditional medicine in many nations, although the mechanism of action is unknown. The hypoglycaemic effects of corn silk are investigated in this review. The phytochemical components present in corn silk-like flavonoids, phenolics, terpenoids, tannins, sterols, and alkaloids are phytochemical components that have hypoglycemic activity and a mechanism for lowering blood glucose levels. There is a lack of a homogenized database on the hypoglycemic properties of corn silk thus the present review attempts to critically analyse it and provide specific recommendations of its doses.

Purification of recombinant bacterial collagens containing structural perturbations.

Streptococcus pyogenes-derived recombinant bacterial collagen-like proteins (CLPs) are emerging as a potential biomaterial for biomedical research and applications. Bacterial CLPs form stable triple helices and lack specific interactions with human cell surface receptors, thus enabling the design of novel biomaterials with specific functional attributes. Bacterial collagens have been instrumental in understanding collagen structure and function in normal and pathological conditions. These proteins can be readily produced in E. coli, purified using affinity chromatography, and subsequently isolated after cleavage of the affinity tag. Trypsin is a widely used protease during this purification step since the triple helix structure is resistant to trypsin digestion. However, the introduction of Gly→X mutations or natural interruptions within CLPs can perturb the triple helix structure, making them susceptible to trypsin digestion. Consequently, removing the affinity tag and isolating collagen-like (CL) domains containing mutations is impossible without degradation of the product. We present an alternative method to isolate CL domains containing Gly→X mutations utilizing a TEV protease cleavage site. Protein expression and purification conditions were optimized for designed protein constructs to achieve high yield and purity. Enzymatic digestion assays demonstrated that CL domains from wild-type CLPs could be isolated by digestion with either trypsin or TEV protease. In contrast, CLPs containing Gly→Arg mutations are readily digested by trypsin while digestion with TEV protease cleaved the His6-tag, enabling the isolation of mutant CL domains. The developed method can be adapted to CLPs containing various new biological sequences to develop multifunctional biomaterials for tissue engineering applications.

Design of a minimal di-nickel hydrogenase peptide.

Ancestral metabolic processes involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13-amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe] hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase, two ancient, extant proteins central to metabolism. These experimental results demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth.

Evaluating Mineral Lattices as Evolutionary Proxies for Metalloprotein Evolution.

Protein coordinated iron-sulfur clusters drive electron flow within metabolic pathways for organisms throughout the tree of life. It is not known how iron-sulfur clusters were first incorporated into proteins. Structural analogies to iron-sulfide minerals present on early Earth, suggest a connection in the evolution of both proteins and minerals. The availability of large protein and mineral crystallographic structure data sets, provides an opportunity to explore co-evolution of proteins and minerals on a large-scale using informatics approaches. However, quantitative comparisons are confounded by the infinite, repeating nature of the mineral lattice, in contrast to metal clusters in proteins, which are finite in size. We address this problem using the Niggli reduction to transform a mineral lattice to a finite, unique structure that when translated reproduces the crystal lattice. Protein and reduced mineral structures were represented as quotient graphs with the edges and nodes corresponding to bonds and atoms, respectively. We developed a graph theory-based method to calculate the maximum common connected edge subgraph (MCCES) between mineral and protein quotient graphs. MCCES can accommodate differences in structural volumes and easily allows additional chemical criteria to be considered when calculating similarity. To account for graph size differences, we use the Tversky similarity index. Using consistent criteria, we found little similarity between putative ancient iron-sulfur protein clusters and iron-sulfur mineral lattices, suggesting these metal sites are not as evolutionarily connected as once thought. We discuss possible evolutionary implications of these findings in addition to suggesting an alternative proxy, mineral surfaces, for better understanding the coevolution of the geosphere and biosphere.

Design of synthetic collagens that assemble into supramolecular banded fibers as a functional biomaterial testbed.

Collagens are the most abundant proteins of the extracellular matrix, and the hierarchical folding and supramolecular assembly of collagens into banded fibers is essential for mediating cell-matrix interactions and tissue mechanics. Collagen extracted from animal tissues is a valuable commodity, but suffers from safety and purity issues, limiting its biomaterials applications. Synthetic collagen biomaterials could address these issues, but their construction requires molecular-level control of folding and supramolecular assembly into ordered banded fibers, comparable to those of natural collagens. Here, we show an innovative class of banded fiber-forming synthetic collagens that recapitulate the morphology and some biological properties of natural collagens. The synthetic collagens comprise a functional-driver module that is flanked by adhesive modules that effectively promote their supramolecular assembly. Multiscale simulations support a plausible molecular-level mechanism of supramolecular assembly, allowing precise design of banded fiber morphology. We also experimentally demonstrate that synthetic fibers stimulate osteoblast differentiation at levels comparable to natural collagen. This work thus deepens understanding of collagen biology and disease by providing a ready source of safe, functional biomaterials that bridge the current gap between the simplicity of peptide biophysical models and the complexity of in vivo animal systems.

Machine learning overcomes human bias in the discovery of self-assembling peptides.

Peptide materials have a wide array of functions, from tissue engineering and surface coatings to catalysis and sensing. Tuning the sequence of amino acids that comprise the peptide modulates peptide functionality, but a small increase in sequence length leads to a dramatic increase in the number of peptide candidates. Traditionally, peptide design is guided by human expertise and intuition and typically yields fewer than ten peptides per study, but these approaches are not easily scalable and are susceptible to human bias. Here we introduce a machine learning workflow-AI-expert-that combines Monte Carlo tree search and random forest with molecular dynamics simulations to develop a fully autonomous computational search engine to discover peptide sequences with high potential for self-assembly. We demonstrate the efficacy of the AI-expert to efficiently search large spaces of tripeptides and pentapeptides. The predictability of AI-expert performs on par or better than our human experts and suggests several non-intuitive sequences with high self-assembly propensity, outlining its potential to overcome human bias and accelerate peptide discovery.

Sucralose hydrogels: Peering into the reactivity of sucralose versus sucrose under lipase catalyzed trans-esterification.

Sucralose differs from sucrose only by virtue of having three Cl groups instead of OH groups. Its intriguing features include being noncaloric, noncariogenic, ∼600 times sweeter than sucrose, stable at high temperatures/acidic pH's, and void of disagreeable aftertastes. These properties are attractive as food additive, one of which is as hydrogel obtainable via the technique of molecular gelation using a sucralose-derived low-molecular weight gelator (LMWG). Such hydrogels are highly responsive to external stimuli like temperature, because the LMWGs self-assemble via non-covalent interactions and could thus be utilized in applications like control-release. We found that sucralose to be unreactive under lipase biocatalysis, unlike sucrose. Hence, the aim of this work was (i) to use computational simulations to further understand sucralose's lack of enzymatic reactivity and (ii) to synthesize the sucralose-based amphiphiles using conventional chemical synthesis and systematically study their tendency towards hydrogelation. Sucrose and sucralose were docked with a high-resolution atomic structure of lipase B from Candida antarctica, modeling the esterification transition state with an active site serine. In extended molecular dynamics simulations, sucrose remained in the active site due to multiple sugar-protein hydrogen bonds. The oxygen-to-chlorine substitutions in sucralose disrupted this hydrogen bonding network. Consistent with observed lack of enzymatic conversion, in multiple simulations, sucralose would rapidly dissociate from the active site. The sucralose-based LMWGs were subsequently synthesized using base-catalyzed conventional chemical synthesis. Three of the sucralose-based amphiphiles (SL-5, SL-6 and SL-7) proved to be successful hydrogelators. The gelators also showed the ability to gel selected beverages. The LMWGs gelled quantities of water and beverage up to 71 and 55 times their weight, respectively, and remain thermally stable up to 144 °C.

Computational design of a sensitive, selective phase-changing sensor protein for the VX nerve agent.

The VX nerve agent is one of the deadliest chemical warfare agents. Specific, sensitive, real-time detection methods for this neurotoxin have not been reported. The creation of proteins that use biological recognition to fulfill these requirements using directed evolution or library screening methods has been hampered because its toxicity makes laboratory experimentation extraordinarily expensive. A pair of VX-binding proteins were designed using a supercharged scaffold that couples a large-scale phase change from unstructured to folded upon ligand binding, enabling fully internal binding sites that present the maximum surface area possible for high affinity and specificity in target recognition. Binding site residues were chosen using a new distributed evolutionary algorithm implementation in protCAD. Both designs detect VX at parts per billion concentrations with high specificity. Computational design of fully buried molecular recognition sites, in combination with supercharged phase-changing chassis proteins, enables the ready development of a new generation of small-molecule biosensors.

Response surface approach to optimize temperature, pH and time on antioxidant properties of wild bush (Plectranthus rugosus) honey from high altitude region (Kashmir Valley) of India.

In this study, the combined effect of temperature (60 to 80 °C) time (10 to 15 min.) and pH (3 to 6) was employed on the anti-oxidant potential (1,1-diphenyl-2-picrylhydrazyl-radical scavenging activity-DPPH-RSA, total phenolic content-TPC, and total flavonoid content-TFC) of wild bush Indian honey from high altitudes of Kashmir Valley by using response surface methodology (RSM). The statistical analysis showed that all the process variables had a substantial effect on the responses related to DPPH-RSA, TFC, and TPC, all of which increased as temperature and time increased. With an increase in pH, the antioxidant activity of wild bush honey was significantly decreased. The heat treatment of honey at high temperature (80 °C) was found to be more efficacious than at 70 and 60 °C, respectively. The findings showed that at higher temperature, browning pigments were formed which enhanced considerably the antioxidant activity of honey.

Quantifying structural relationships of metal-binding sites suggests origins of biological electron transfer.

Biological redox reactions drive planetary biogeochemical cycles. Using a novel, structure-guided sequence analysis of proteins, we explored the patterns of evolution of enzymes responsible for these reactions. Our analysis reveals that the folds that bind transition metal­containing ligands have similar structural geometry and amino acid sequences across the full diversity of proteins. Similarity across folds reflects the availability of key transition metals over geological time and strongly suggests that transition metal­ligand binding had a small number of common peptide origins. We observe that structures central to our similarity network come primarily from oxidoreductases, suggesting that ancestral peptides may have also facilitated electron transfer reactions. Last, our results reveal that the earliest biologically functional peptides were likely available before the assembly of fully functional protein domains over 3.8 billion years ago.Thus, life is a special, very complex form of motion of matter, but this form did not always exist, and it is not separated from inorganic nature by an impassable abyss; rather, it arose from inorganic nature as a new property in the process of evolution of the world. We must study the history of this evolution if we want to solve the problem of the origin of life. [A. I. Oparin (1)]

Biophysical Characterization of Iron-Sulfur Proteins.

Iron-sulfur proteins are primordial catalysts and biological electron carriers that today drive major metabolic pathways across all forms of life. They can access a diversity of oxidation states and can mediate electron transfer over an extended range of reduction potentials spanning more than 1 V. Depending on the protein micro-environment and geometry of ligand, co-ordination the iron-sulfur clusters can occur in different forms [2Fe-2S], [3Fe-4S], HiPIP [4Fe-4S], and [4Fe-4S]. There are several spectroscopic methods available to characterize the composition and electronic configuration of the iron-sulfur clusters, such as optical methods and electron paramagnetic resonance. This paper presents the protocols used to characterize the metal center of Coiled-Coil Iron-Sulfur (CCIS), an artificial metalloprotein containing one [4Fe-4S] cluster. It is expected that these protocols will be of general utility for other iron-sulfur proteins.

A Folding Insulator Defines Cryptic Domains in Tropomyosin.

Multidomain proteins are the product of evolutionary selection for diversity of function through concatenation and repurposing of existing modular units of structures. In structures of proteins with multiple domains, components are often globular units stitched together with flexible linkers. Multidomain proteins often fold as multiple distinct order-disorder transitions. However, the relationship between structure and folding is not always straightforward. Tropomyosin binds to actin in muscle and cytoskeletal filaments. The structure is that of a continuous ɑ-helix lacking domain boundaries, but unfolding shows distinct transitions suggesting at least three possible domains do exist. To explore how domains might occur in a continuous structure, we used Lifson-Roig helix-coil models with sequence domains of varying helical nucleation propensities. Of these models, ones with a central folding insulator, separating folding of N- and C-terminal domains, are most consistent with experimental folding studies. The positions of domain boundaries are identified by hydrogen-deuterium exchange mass spectrometry. The presence of structurally cryptic folding domains in tropomyosin could relate to its evolution and explain the uneven distribution of deleterious mutations that lead to various cardiomyopathies.

Anaerobic Expression and Purification of Holo-CCIS, an Artificial Iron-sulfur Protein.

Iron-sulfur proteins are ubiquitous among all living organisms and are indispensable for almost all metabolic pathways ranging from photosynthesis, respiration, nitrogen, and carbon dioxide cycles. The iron-sulfur clusters primarily serve as electron acceptors and donors and transfer electrons to active sites of various enzymes, thus driving the energy metabolism. Prokaryotes like E. coli have ISC and SUF pathways that help in the assembly and maturation of iron-sulfur proteins. These iron-sulfur proteins, especially with [4Fe-4S] clusters, are highly sensitive to molecular oxygen, and it would be advantageous if the de novo proteins and native proteins having iron-sulfur binding sites are expressed and isolated under anaerobic conditions. Bacterially assembled iron-sulfur proteins, when isolated and purified anaerobically, exhibit improved biochemical and biophysical stabilities in comparison to the counterparts expressed and purified aerobically and reconstituted under anaerobic conditions. This protocol outlines the expression and purification of the artificial protein, Coiled-Coil Iron-Sulfur (CCIS). It may be deployed to both natural and artificial [4Fe-4S] proteins when heterologously expressed in E. coli.