Hydrophobic Protein Research Articles

Protein N-terminal acylation: An emerging field in bacterial cell physiology.

N-terminal (Nt)-acylation is the irreversible addition of an acyl moiety to the terminal alpha amino group of a peptide chain. This type of modification alters the nature of the N terminus, which can interfere with the function of the modified protein by disrupting protein interactions, function, localization, degradation, hydrophobicity, or charge. Nt acylation is found in all domains of life and is a highly common occurrence in eukaryotic cells. However, in prokaryotes very few cases of Nt acylation have been reported. It was once thought that Nt acylation of proteins, other than ribosomal proteins, was uncommon in prokaryotes, but recent evidence suggests that this modification may be more common than once realized. In this review, we discuss what is known about prokaryotic Nt acetylation and the acetyltransferases that are responsible, as well as recent advancements in this field and currently used methods to study Nt acetylation.

Metal tolerance gene family in barley: an in silico comprehensive analysis

Metal-tolerance proteins (MTPs) are divalent cation transporters that play critical roles in metal tolerance and ion homeostasis in plants. However, a comprehensive study of MTPs is still lacking in crop plants. The current study aimed to comprehensively identify and characterize the MTP gene family in barley (Hordeum vulgare, Hv), an important crop. In total, 12 HvMTPs were identified in the barley genome in this study. They were divided into three phylogenetic groups (Zn-cation diffusion facilitator proteins [CDFs], Fe/Zn-CDFs, and Mn-CDFs) and further subdivided into seven groups (G1, G5, G6, G7, G8, G9, and G12). The majority of MTPs were hydrophobic proteins found in the vacuolar membrane. Gene duplication analysis of HvMTPs revealed one pair of segmental-like duplications in the barley genome. Evolutionary analysis suggested that barley MTPs underwent purifying natural selection. Additionally, the HvMTPs were analyzed in the pan-genome sequences of barley (20 accessions), which suggests that HvMTPs are highly conserved in barley evolution. Cis-acting regulatory elements, microRNA target sites, and protein–protein interaction analysis indicated the role of HvMTPs in a variety of biological processes. Expression profiling suggests that HvMTPs play an active role in maintaining barley nutrient homeostasis throughout its life cycle, and their expression levels were not significantly altered by abiotic stresses like cold, drought, or heat. The expression of barley HvMTP genes in the presence of heavy metals such as Zn2+, Cu2+, As3+, and Cd2+ revealed that these MTPs were induced by at least one metal ion, implying their involvement in metal tolerance or transportation. The identification and comprehensive investigation of MTP gene family members will provide important gene resources for the genetic improvement of crops for metal tolerance, bioremediation, or biofortification of staple crops.

Microalgal extract as bio-coating to enhance biofilm growth of marine microalgae on microporous membranes

Microalgal biofilm is a popular platform for algal production, nutrient removal and carbon capture; however, it suffers from significant biofilm exfoliation under shear force exposure. Hence, a biologically-safe coating made up of algal extracellular polymeric substances (EPS) was utilized to secure the biofilm cell retention and cell loading on commercial microporous membrane (polyvinylidene fluoride), making the surfaces more hydrophobic (contact angle increase up to 12°). Results demonstrated that initial cell adhesion of three marine microalgae (Amphora coffeaeformis, Cylindrotheca fusiformis and Navicula incerta) was enhanced by at least 1.3 times higher than that of pristine control within only seven days with minimized biofilm exfoliation issue due to uniform distribution of sticky transparent exopolymer particles. Bounded extracellular polysaccharide gathered was approximately 23% higher on EPS-coated membranes to improve the biofilm's hydraulic resistance, whereas bounded extracellular protein would only be substantially elevated after the attached cells re-accommodate themselves onto the EPS pre-coating of themselves. In accounting the rises of hydrophobic protein content, biofilm was believed to be more stabilized, presumably via hydrophobic interactions. EPS biocoating would generate a groundswell of interest for bioprocess intensifications though there are lots of inherent technical and molecular challenges to be further investigated in future.

Correction to: P343 Novel hydrophobic binding surface proteins are instrumental for phagocytosis of Lichtheimia corymbifera by macrophages.

Correction to: P343 Novel hydrophobic binding surface proteins are instrumental for phagocytosis of Lichtheimia corymbifera by macrophages.

Intestinal Aquaporins

Aquaporins (AQPs) are integral, hydrophobic, and transmembrane proteins that facilitate passive transport of water depending on the osmotic pressure on both sides of the cell membrane. Of these channel proteins, those that are permeable only to water are called 'classical aquaporins', those that allow the passage of small molecules such as glycerol in addition to water are called 'aquaglyceroporins', and those with different structures and cellular distributions are called 'super aquaporins'. Aquaporins have a great role in the gastrointestinal tract as well as in the whole organism. Transepithelial transport of fluid in the intestine occurs spontaneously, either by paracellular or cellular routes, or both. The paracellular pathway is mediated by tight junctions in the intestinal epithelium and their passage is regulated based on the size and load of substances, while the cellular pathway is passive diffusion mediated by aquaporins and co-transporters. Among them, aquaporins are the major cellular pathway for bidirectional fluid transport in the gut. Aquaporins have important roles in the gut. Based on these roles, information, and research on whether aquaporins can be regulated by drugs and dietary supplements to increase intestinal health and improve their functions is increasing day by day. In this review, the functions of aquaporins in the intestinal tract, their situations in intestinal diseases, and the drugs and dietary supplements used for the treatment of these diseases are discussed together with current studies.

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli

Expression of affinity-tagged recombinant proteins for crystallography, protein–protein interaction, antibody generation, therapeutic applications, etc. mandates the generation of high-yield soluble proteins. Although recent developments suggest the use of yeast, insect, and mammalian cell lines as protein expression platforms, Escherichia coli is still the most popular, due mainly to its ease of growth, feasibility in genetic manipulation and economy. However, some proteins have a spontaneous tendency to form inclusion bodies (IBs) when over-expressed in bacterial expression systems such as E. coli, thus posing a challenge in purification and yield. At times, small peptides undergo degradation during protein production and hence using suitable tags could circumvent the problem. Although several independent techniques have been used to solubilize IBs, these cannot always be applied in a generic sense. Although tagging a GST moiety is known to enhance the solubility of fusion proteins in E. coli, resulting in yields of 10–50 mg/L of the culture, the inherent nature of the protein sequence at times could lead to the formation of IBs. We have been working on a Myc Binding Protein-1 orthologue, viz. Flagellar Associated Protein 174 (FAP174) from the axoneme of Chlamydomonas reinhardtii that binds to an A-Kinase Anchoring Protein 240 (AKAP240) which has been annotated as Flagellar Associated Protein 65 (FAP65). Using an in-silico approach, we have identified two amphipathic helices on FAP65 (CrFAP65AH1 and CrFAP65AH2) that are predicted to bind to FAP174. To test this prediction, we have cloned the GST-tagged peptides, and overexpressed them in E. coli that have resulted in insoluble IBs. The yields of these over-expressed recombinant proteins dropped considerably due to IB formation, indicating aggregation. An integrated approach has been used to solubilize four highly hydrophobic polypeptides, viz. two amphipathic helices and the respective proline variants of FAP65. For solubilizing these polypeptides, variables such as non-denaturing detergents (IGEPAL CA-630), changing the ionic strength of the cell lysis and solubilization buffer, addition of BugBuster®, diluting the cell lysate and sonication were introduced. Our statistically viable results yielded highly soluble and functional polypeptides, indiscreet secondary structures, and a yield of ~ 20 mg/L of the E. coli culture. Our combinatorial strategy using chemical and physical methods to solubilize IBs could prove useful for hydrophobic peptides and proteins with amphipathic helices.

Cloning, subcellular localization and expression analysis of squalene epoxidase gene BsSE1 from Bletilla striata

Squalene epoxidase catalyzes the oxidation of squalene to 2,3-oxo-squalene (BsSE1), and is the key rate limiting enzyme in the synthesis of triterpenoids and sterols in plants. This study focused on the basic aspects of BsSE1 including the sequence information, sub-cellular localization expression patterns of BsSE1. Using to the sequence information of Bletilla striata transcriptome, the full-length CDS of BsSE1 gene was amplified. The physicochemical properties and structural characteristics of BsSE1 protein were analyzed by bioinformatics analysis software, and vector was constructed to analyze the protein locations and expression patterns. The results showed that the CDS of BsSE1 gene was 1542 bp, encoding 513 amino acids. BsSE1 protein is a hydrophobic protein with two transmembrane domains but no signal peptides. It is localied in the endoplasmic reticulum membrane and belongs to the typical squalene epoxidase gene. BsSE1 has the closest genetic relationship with SE protein of Dendrobium officinale and Phalaenopsis equestris. The expression level of BsSE1 was higher in pseudobulblet of Bletilla striata seedlings, followed by roots, and lower in seedling stems. After SA induction, the expression of BsSE1 in Bletilla striata showed significant changes, increased first, then decreased, finally increase again. The results provide a basis for further study of this gene family in plants.

Ohmic baking of gluten‐free bread: role of non‐gluten protein on GF bread structure and properties

SummaryBatter stabilisation presents a great challenge for gluten‐free (GF) bread, as CO2 is released during bread‐making process, resulting in small, dense and crumbly breads. Apart from starch, protein plays a crucial role in gas cell stabilisation. This study aims to assess the effect of non‐gluten protein from different sources (plant and animal) on GF batter's rheological behaviour (pasting properties, rheology and foam stability) and on bread properties after baking with conventional and ohmic heating method. Hence, this study evaluated the functional properties (protein solubility, hydrophobicity, sulfhydryl groups, foaming and emulsification properties) of selected non‐gluten protein relevant for foam stabilisation. Furthermore, a correlation matrix was established by involving the functional properties of the proteins and their interaction with starch on batter rheology and bread quality. Among proteins, egg albumin and potato proteins were reported to perform superior functionality in GF bread; in particular, potato protein generated breads with the highest volume for both baking methods, which was potential to replace egg albumin. According to the correlation matrix, protein solubility was required in foaming and emulsification behaviour to improve GF bread properties.

Interaction between Movement Proteins of Hibiscus green spot virus.

Movement proteins (MPs) of plant viruses enable the translocation of viral genomes from infected to healthy cells through plasmodesmata (PD). The MPs functions involve the increase of the PD permeability and routing of viral genome both to the PD entrance and through the modified PD. Hibiscus green spot virus encodes two MPs, termed BMB1 and BMB2, which act in concert to accomplish virus cell-to-cell transport. BMB1, representing an NTPase/helicase domain-containing RNA-binding protein, localizes to the cytoplasm and the nucleoplasm. BMB2 is a small hydrophobic protein that interacts with the endoplasmic reticulum (ER) membranes and induces local constrictions of the ER tubules. In plant cells, BMB2 localizes to PD-associated membrane bodies (PAMBs) consisting of modified ER tubules and directs BMB1 to PAMBs. Here, we demonstrate that BMB1 and BMB2 interact in vitro and in vivo, and that their specific interaction is essential for BMB2-directed targeting of BMB1 to PAMBs. Using mutagenesis, we show that the interaction involves the C-terminal BMB1 region and the N-terminal region of BMB2.

Changes in the microbiota of a vacuum-packed cooked bass product and the effects of cobalt irradiation on its quality during storage

This study investigates changes in the microbiota of vacuum-packed cooked bass (Micropterus salmoides), including the effects of different doses of 60Co-γ irradiation (0, 1, 3, and 5 kGy) on muscle quality, during storage at 4 °C (0–15 days). Analyses for other indicators like the total bacterial colony counts and myofibrillar protein concentration were carried out. 16SrDNA high-throughput sequencing results showed that Stenotrophomonas maltophilia had the highest relative abundance in bass after storage. Additionally, the total bacterial colony counts and total volatile basic nitrogen (TVB-N) content of the irradiated samples were within the standard limits after day 15. During 0–6 days of storage, 3 kGy irradiation increased the myofibrillar protein concentration and the water-holding capacity of the fish compared with other doses. However, 3 kGy irradiation had less influence on the surface hydrophobicity of myofibrillar proteins than 5 kGy irradiation. Overall, Stenotrophomonas maltophilia was the dominant spoilage bacteria in bass, and 3 kGy irradiation treatment effectively inhibited microbial proliferation and muscle quality deterioration during storage at 4 °C. Moreover, the optimal storage time for bass products after 3 kGy irradiation treatment was 6 days. This provides a basis for improving bass quality during storage and promoting the application of irradiation technology.

Impact of Different Frozen Dough Technology on the Quality and Gluten Structure of Steamed Buns

To advance the industrialization production of steamed buns, the current study explored the freeze-stability of unfermented, pre-fermented and par-steamed frozen dough. The results showed that the steamed bun made from unfermented dough with 2.0% yeast, the pre-fermented dough with a pre-fermented time of 30 min and the par-steamed dough with a pre-steamed time of 15 min showed the best sensory properties quality upon frozen storage. The gassing power of un- and pre-fermented dough gradually decreased, and dough with longer pre-fermented time exhibited more evident loss of gassing power. Freeze-induced depolymerization of gluten protein was the least distinct in the par-steamed dough, followed by the pre- and un-fermented dough, which was probably related to the superior freeze stability of glutenin-gliadin macro-crosslinks upon the pre-steaming stage. The surface hydrophobicity of gluten proteins of frozen dough decreased during the initial storage and was enhanced subsequently, which was related with the combined effects of the unfolding and synchronous aggregation induced by freezing and steaming, respectively. Moreover, the surface hydrophobicity of gluten in par-steamed frozen dough and steamed buns was more resistant to frozen storage, which was probably attributed to the established stable structure during the pre-steaming process.

Cloning, Expression Analysis, and Functional Characterization of Candidate Oxalate Transporter Genes of HbOT1 and HbOT2 from Rubber Tree (Hevea brasiliensis)

Secretion of oxalic acid from roots is an important aluminum detoxification mechanism for many plants such as Hevea brasiliensis (rubber tree). However, the underlying molecular mechanism and oxalate transporter genes in plants have not yet been reported. In this study, the oxalate transporter candidate genes HbOT1 and HbOT2 from the rubber tree were cloned and preliminarily identified. It was found that HbOT1 had a full length of 1163 bp with CDS size of 792 bp, encoding 263 amino acids, and HbOT2 had a full length of 1647 bp with a CDS region length of 840 bp, encoding 279 amino acid residues. HbOT1 and HbOT2 were both stable hydrophobic proteins with transmembrane structure and SNARE_assoc domains, possibly belonging to the SNARE_assoc subfamily proteins of the SNARE superfamily. qRT-PCR assays revealed that HbOT1 and HbOT2 were constitutively expressed in different tissues, with HbOT1 highly expressed in roots, stems, barks, and latex, while HbOT2 was highly expressed in latex. In addition, the expressions of HbOT1 and HbOT2 were up-regulated in response to aluminum stress, and they were inducible by metals, such as copper and manganese. Heterologous expression of HbOT1 and HbOT2 in the yeast mutant AD12345678 enhanced the tolerance to oxalic acid and high concentration aluminum stress, which was closely correlated with the secretion of oxalic acid. This study is the first report on oxalate transporter genes in plants, which provides a theoretical reference for the study on the molecular mechanism of oxalic acid secretion to relieve aluminum toxicity and on aluminum-tolerance genetic engineering breeding.

Effects of hexane on protein profile and techno-functional properties of pea protein isolates

The defatting of legume flours with hexane is usually the first step in producing protein-rich ingredients. However, its impact on protein profiles, zeta potential, surface hydrophobicity and techno-functionality of pea proteins has not been evaluated. Consequently, this work aimed to evaluate the impact of the hexane defatting step on pea protein profiles, surface hydrophobicity and zeta potential, as well as techno-functional properties of non-defatted and defatted pea protein isolates. The results showed that alkaline extraction of hexane-defatted pea flour increased the net surface charge (zeta-potential) and reduced particle size of the pea protein isolate. Moreover, only the foaming properties of pea protein isolate generated from defatted pea flour were improved. Consequently, except for improving foaming properties, the defatting step is not essential for the production of pea protein isolate.

Valorizing protein-polysaccharide conjugates from sugar beet pulp as an emulsifier

Inspired by the emulsion stability of sugar beet pulp pectin, the hydrophobic protein fraction in sugar beet pulp (SBP) is expected to feature high interfacial activity. This work retrieved alkaline extracted protein-polysaccharide conjugates (AEC) from partially depectinized SBP by hot alkaline extraction. AEC was protein-rich (57.20 %), and the polysaccharide mainly comprised neutral sugar, which adopted a rhamnogalacturonan-I pectin-like structure. The hydrophobic polypeptide chains tangled as a dense ‘core’ with polysaccharide chains attached as a hydrated ‘shell’ (hydrodynamic radius of ~110 nm). AEC could significantly decrease the oil-water interfacial tension (11.58 mN/m), featuring superior emulsification performance than three control emulsifiers, especially the excellent emulsifying stability (10 % oil) as the emulsion droplet size of 0.438 and 0.479 μm for fresh and stored (60 °C, 5 d) emulsions, respectively. The relationship of molecular structure to emulsification was investigated by specific enzymic modification, suggesting the intact macromolecular structure was closely related to emulsifying activity and that the NS fraction contributed greatly to emulsifying stability. Moreover, AEC was highly efficient to stabilize gel-like high internal phase emulsions (oil fraction 0.80) with low concentration (0.2 %) and even high ionic strength (0–1000 mM). Altogether, valorizing AEC as an emulsifier is feasible for high-value utilization of SBP.

Effect of two-stage heating treatment on physicochemical properties of shrimp (Penaeus vannamei) meat.

Changes in the physicochemical properties of shrimp meat treated with two-stage heating were investigated. Currently, shrimp products in the processing process are susceptible to uneven dehydration, shrimp meat shrinkage, which results in rough and hard texture, poor chewiness, and seriously affects the edible quality as well as economic benefits. Improving the utilization value of shrimp resources, expanding its market shares, optimizing the tenderness of shrimp is the key to developing new types of fresh and ready-to-eat shrimp products. The results indicated that preheating at 30 °C could not affect the quality of shrimp meat significantly (P > 0.05). As the preheating temperature increased from 40 °C to 50 °C, the hardness and shear force of shrimp meat decreased due to the exposure of protein hydrophobic groups, protein aggregation and degradation, muscle fraction broken, and weight loss increase. Further increase in preheating temperature would lead to further aggregation and gelation of proteins, causing hardness and shear force increase. Besides, the results of microstructure showed that preheating at 40 °C and 50 °C could cause the shrimp muscles to become loose. This study showed that the preheating temperature ranging from 40 °C to 50 °C could effectively improve the tenderness of shrimp meat. This study might be useful for developing tenderized shrimp products in the future. © 2022 Society of Chemical Industry.

Learning the relationship between nanoscale chemical patterning and hydrophobicity

The hydrophobicity of proteins and similar surfaces, which display chemical heterogeneity at the nanoscale, drives countless aqueous interactions and assemblies. However, predicting how surface chemical patterning influences hydrophobicity remains a challenge. Here, we address this challenge by using molecular simulations and machine learning to characterize and model the hydrophobicity of a diverse library of patterned surfaces, spanning a wide range of sizes, shapes, and chemical compositions. We find that simple models, based only on polar content, are inaccurate, whereas complex neural network models are accurate but challenging to interpret. However, by systematically incorporating chemical correlations between surface groups into our models, we are able to construct a series of minimal models of hydrophobicity, which are both accurate and interpretable. Our models highlight that the number of proximal polar groups is a key determinant of hydrophobicity and that polar neighbors enhance hydrophobicity. Although our minimal models are trained on particular patch size and shape, their interpretability enables us to generalize them to rectangular patches of all shapes and sizes. We also demonstrate how our models can be used to predict hot-spot locations with the largest marginal contributions to hydrophobicity and to design chemical patterns that have a fixed polar content but vary widely in their hydrophobicity. Our data-driven models and the principles they furnish for modulating hydrophobicity could facilitate the design of novel materials and engineered proteins with stronger interactions or enhanced solubilities.

Oxidation affects dye binding of myofibrillar proteins via alteration in net charges mediated by a reduction in isoelectric point

In order to study the effect of oxidation on the dye-binding behavior of myofibrillar proteins, selected dyes with different charges (positively charged Sarfarin O (SO), neutral bromophenol blue (BPB), and negatively charged Orange G (OG)) were incubated with myofibrils oxidized by the Fenton system with H2O2 (10 mM). Upon oxidation, loss of free thiols, formation of carbonyls, particle size, and hydrophobicity of myofibrillar proteins (MPs) increased. The absolute value of Zeta-potential increased by 14.48 % after oxidation, the myofibrillar proteins shifted to a more acidic isoelectric point (pI) upon oxidation. Oxidation decreased net positive charges of myofibrillar protein and the binding ability of MPs towards OG in the environment with pH less than pI and the affinity of MPs towards SO in the environment with pH more than pI were thus increased. Here we propose a hypothesis that oxidation-induced change in net charges is the driving force affecting the amount of protein-bound dye. This paper aims to examine the effect of oxidation on the net charges of myofibrillar proteins and to provide insight into the mechanism of oxidation-induced changes in protein-bound dyes.

Influence of LAB Fermentation on the Color Stability and Oxidative Changes in Dry-Cured Meat

Consumption of food products with a high level of compounds that are products of fat or protein oxidation is associated with the onset of various diseases. Therefore, this study estimated the level of oxidation in a fermented long-maturing tenderloin inoculated with lactic acid bacteria strains. To estimate the level of fat and protein exposure to oxidative factors, thiobarbituric acid reactive substances (TBARS), oxidation–reduction potential (ORP), oxygenation index (Oxi) as well as surface hydrophobicity (HS) of protein, and the content of free sulfhydryl (SH) groups were used as indicators of oxidation status. To validate the results, changes in the color of the products were determined using instrumental methods. This study confirmed the relationship between fat oxidation (based on TBARS) and oxidation of myoglobin as a protein representative (based on Oxi). Indeed, statistical analysis showed that TBARS, Oxi and SH were correlated with each other and could be used as indicators of oxidation in fermented products. The findings of this study also showed the use of LAB as starter cultures for meat fermentation may have an impact on the level of oxidation; in particular, the BAUER strain showed a protective effect on proteins in the final stages of the production of dry-cured pork loins.

Effect of Alcalase Modification of Yellow Lupin (Lupinus luteus L.) Protein Isolate on Some Functional Properties and Antioxidant Activity.

Protein isolate obtained from sweet yellow lupin (Lupinus luteus L.) and its Alcalase hydrolysates were examined for their functional and antioxidant properties in relation to surface hydrophobicity of proteins and peptides and molecular weight distribution. Enzymatic hydrolysis improved the foaming characteristics of lupin proteins, while the emulsifying properties deteriorated. It means that good foaming properties of preparations are determined by the presence of low-molecular δ conglutin and small subunits of γ conglutins. In turn, larger proteins such as α and β conglutin are responsible for maintaining good emulsifying properties. The measured surface hydrophobicity was consistent with the results of emulsifying properties. It has also been shown that the scope of changes in antioxidant properties due to hydrolysis, measured by DPPH method and as reducing power, is more pronounced than with the use of ABTS and FRAP methods.

Overexpression of RuFLS2 Enhances Flavonol-Related Substance Contents and Gene Expression Levels.

As an emerging third-generation fruit, blackberry has high nutritional value and is rich in polyphenols, flavonoids and anthocyanins. Flavonoid biosynthesis and metabolism is a popular research topic, but no related details have been reported for blackberry. Based on previous transcriptome data from this research group, two blackberry flavonol synthase genes were identified in this study, and the encoded proteins were subjected to bioinformatics analysis. RuFLS1 and RuFLS2 are both hydrophobic acidic proteins belonging to the 2OG-Fe(II) dioxygenase superfamily. RuFLS2 was expressed at 27.93-fold higher levels than RuFLS1 in red-purple fruit by RNA-seq analysis. Therefore, RuFLS2-overexpressing tobacco was selected for functional exploration. The identification of metabolites from transgenic tobacco showed significantly increased contents of flavonoids, such as apigenin 7-glucoside, kaempferol 3-O-rutinoside, astragalin, and quercitrin. The high expression of RuFLS2 also upregulated the expression levels of NtF3H and NtFLS in transgenic tobacco. The results indicate that RuFLS2 is an important functional gene regulating flavonoid biosynthesis and provides an important reference for revealing the molecular mechanism of flavonoid accumulation in blackberry fruit.