Changes In Protein Secondary Structure Research Articles

Raman spectroscopy in the study of amyloid formation and phase separation.

Neurodegenerative diseases, such as Alzheimer's and Parkinson's, share a common pathological feature of amyloid structure accumulation. However, the structure-function relationship between these well-ordered, β-sheet-rich, filamentous protein deposits and disease etiology remains to be defined. Recently, an emerging hypothesis has linked phase separation, a process involved in the formation of protein condensates, to amyloid formation, suggesting that liquid protein droplets serve as loci for amyloid initiation. To elucidate how these processes contribute to disease progression, tools that can directly report on protein secondary structural changes are needed. Here, we review recent studies that have demonstrated Raman spectroscopy as a powerful vibrational technique for interrogating amyloid structures; one that offers sensitivity from the global secondary structural level to specific residues. This probe-free technique is further enhanced via coupling to a microscope, which affords structural data with spatial resolution, known as Raman spectral imaging (RSI). In vitro and in cellulo applications of RSI are discussed, highlighting studies of protein droplet aging, cellular internalization of fibrils, and Raman imaging of intracellular water. Collectively, utilization of the myriad Raman spectroscopic methods will contribute to a deeper understanding of protein conformational dynamics in the complex cellular milieu and offer potential clinical diagnostic capabilities for protein misfolding and aggregation processes in disease states.

Synthesis of hydroxyethyl starch 200/0.5-loaded albumin nanoparticles: biocompatibility and interaction mechanism.

We aimed to synthesize hydroxyethyl starch (HES) 200/0.5-loaded bovine serum albumin nanoparticles (HBNs) and investigate the compatibility and binding mechanism in simulated physiological environments. Here, to elucidate the morphology, biocompatibility, and formation mechanism of HBNs, techniques such as scanning electron microscopy, haemolysis test, fluorescence, and circular dichroism spectroscopy were applied. The thermodynamic parameters at body temperature (ΔS° = -26.7J·mol-1 ·K-1 , ΔH° = -3.20 × 104 J·mol-1 , and ΔG = -2.35 × 104 J·mol-1 ) showed a 1:1 binding stoichiometry, which was formed by hydrogen bonds and van der Waals interactions. In addition, the conformational analysis showed that the microenvironment of fluorophores was altered with the adaptational protein secondary structural changes. Energy transfer occurred from the fluorophores to HES with a high possibility. All these results provided accurate and complete primary data for demonstrating the interaction mechanisms of HES with BSA, which helps to understand its pharmaceutical effects in blood.

Molecular Evidence of Structural Changes in Silk Using Unlimited Degradation Mass Spectrometry.

Proteomics has important uses in archeological science because it can distinguish species, reveal the evolution of paleontology, and provide biological evidence of historical events. However, this technique still has full potential in the study of silk aging mechanisms. In this work, we propose a strategy combining unlimited degradation with mass-spectrometry-based proteomics techniques, which interpret protein fragmentation propensity and secondary structure changes by detecting content changes of specific peptide groups in complex proteomes. This approach was employed to study the conformational changes in silk microscopic crystals after heat treatment. Combining conventional mechanics and crystallographic characterization, a thermal aging degradation mechanism model was proposed. At the same time, it explained the interesting problem that the crystallinity remained unchanged, but the mechanical properties decreased significantly. Focusing on the unlimited degradation process, this method will be widely applicable to the study of silk and wool aging processes and regenerated silk fibroin.

Effects of different ohmic heating treatments on parvalbumin structure and reduction of allergenicity in Japanese eel (Anguilla japonica)

We investigated the effects of ohmic heating (OH) on the structural properties and allergenicity of parvalbumin (PV). Compared to other heating methods (water bath heating (WH), OH combined with WH, and OH combined with air thermostatic heating (AH)), pure OH heating expended the least time and total energy. PV sensitization was reduced by approximately 65% by pure OH heating. SDS-PAGE, tricine–SDS-PAGE, and western blotting analyses revealed a molecular weight of sensitized β-PV of about 12 kDa. Band intensity decreased with increasing OH time, and significant changes were observed in amino acid content, secondary structure, microstructure, and dielectric properties. Reducing PV, allergenicity through protein unfolding and secondary structural changes, thereby possibly reducing the allergenicity of eel, provides a theoretical basis for developing hypoallergenic products.

Effects of chickpea protein-stabilized Pickering emulsion on the structure and gelling properties of hairtail fish myosin gel.

The effects of different amount (0-12%) of chickpea protein-stabilized Pickering emulsion (CPE) on the gelling properties, intermolecular interactions, microstructure, and physicochemical stability of hairtail fish myosin gels were investigated. The myosin gel with 6%-9% CPE demonstrated significantly higher viscoelasticity, gel strength, hardness, water-holding capacity and whiteness, compared to the control (P<0.05). In addition, Raman spectroscopy showed that CPE changed the microenvironment of the myosin, which promoted the changes in protein secondary structures, disulfide bond conformation and the local environments of the composite gels. The addition of 6%-9% CPE also enhanced the disulfide bond and hydrophobic interaction of myosin gels which induced more compact gel network structures. Furthermore, CPE improved the lipid oxidative stability and freeze-thaw stability of myosin gel. The results indicated that CPE could improve the gelling properties of myosin, making it a potential new additive and lipid substitute for the development of new emulsion gel products.

Variations in the Protein Hydration and Hydrogen-Bond Network of Water Molecules Induced by the Changes in the Secondary Structures of Proteins Studied through Near-Infrared Spectroscopy.

This study investigated how the secondary structural changes of proteins in aqueous solutions affect their hydration and the hydrogen-bond network of water molecules using near-infrared (NIR) spectroscopy. The aqueous solutions of three types of proteins, i.e., ovalbumin, β-lactoglobulin, and bovine serum albumin, were denatured by heating, and changes in the NIR bands of water reflecting the states of hydrogen bonds induced via protein secondary structural changes were investigated. On heating, the intermolecular hydrogen bonds between water molecules as well as between water and protein molecules were broken, and protein molecules were no longer strongly bound by the surrounding water molecules. Consequently, the denaturation was observed to proceed depending on the thermodynamic properties of the proteins. When the aqueous solutions of proteins were cooled after denaturation, the hydrogen-bond network was reformed. However, the state of protein hydration was changed owing to the secondary structural changes of proteins, and the variation patterns were different depending on the protein species. These changes in protein hydration may be derived from the differences in the surface charges of proteins. The elucidation of the mechanism of protein hydration and the formation of the hydrogen-bond network of water molecules will afford a comprehensive understanding of the protein functioning and dysfunctioning derived from the structural changes in proteins.

Extraction and modification of protein from sesame oil cake by the application of emerging technologies

Present work evaluated the extraction yield and quality of protein from defatted sesame meal (DSM) using ultrasound-assisted extraction (UAE), low-temperature microwave-assisted extraction (MAE), and moderate electric field-assisted extraction (MEFAE). It was observed that the application of emerging technologies as pre-treatments significantly increased protein yield from 26.4% (without pre-treatments) to 38.9% (with UAE 450 W–20 min), 36.05% for MAE (with 500 W–20 min) and 32.75% for MEFAE (with 100 V–30 min) and improved the functional properties of Sesame protein isolate (SPI). FTIR revealed Amide I to III bands in SPI, with characteristic changes in protein secondary structure. The treated sample showed higher thermal stability evident from the denaturation temperature increase from 203.41 to 221.40 °C. The protein's microstructure in this study was transformed using various extraction techniques into a compact structure with a wrinkled surface. MAE-SPI showed maximum Invitro protein digestibility as 94.44%. The molecular weight profile revealed that the SPI samples' protein bands had molecular weights between 6.5 and 200 kDa. SDS-PAGE results revealed a change in the intensity of protein bands, and all of the main allergen protein bands were degraded.

Lutein fortification of wheat bread with marigold powder: Impact on rheology, water dynamics, and structure.

Demands for foods conducive to eye health have been on the increase in the global healthcare sector. Marigold powder as a major source of lutein was utilized to produce lutein-fortified breads for ocular health. The physicochemical characteristics of the doughs and breads were investigated in terms of rheology, water mobility, and protein secondary structures. The incorporation of marigold powder decreased the water absorption of doughs without significantly altering thermo-mechanical properties. With a range of fortification levels (1 ~ 3%), marigold powder led to decreased storage and loss modulus of doughs by weakening their gluten network, which was supported by their T2 relaxation times. The resistances of the doughs weakened with increasing levels of marigold powder, while their extensibilities significantly incremented. FTIR spectral deconvolution revealed the changes of wheat protein structures upon marigold powder incorporation, in which the proportion of β-turn increased at the expense of β-sheet ratio. The breads with marigold powder displayed increased specific volume from 4.034 to 4.368 mL/g, accompanied by softer textures. The baking process led to heat-induced losses in lutein concentration of less than 10% within the crumbs and approximately 30% in the crusts. The use of marigold powder induced changes in protein secondary structure and extensional features of doughs, contributing to increased loaf volume and softer texture. Overall, this study provides fundamental information about the rheological and structural effects of marigold powder in a wheat bread system, consequently encouraging the food industry to utilize marigold power as a functional food ingredient. This article is protected by copyright. All rights reserved.

Abstract 5561: Molecular imaging of calcification and soft tissue defines prognosis of DCIS to invasive breast cancer

Abstract Introduction: Ductal carcinoma in situ (DCIS) is a non-obligatory precursor to invasive breast cancer (IBC). Currently there are no reliable markers to indicate if DCIS will progress IBC after an initial treatment, or if it will remain as DCIS. Because of this uncertainty, standard treatment for DCIS includes surgery with or without radiotherapy. Breast microcalcifications are a common feature in the mammographic detection of DCIS and occur in around 80% of DCIS and 50% of IBC. Despite this, their potential role in the development of DCIS and with progression to IBC remains largely unexplored. The main aim of the study is to identify novel biomarkers for DCIS prognosis, based on chemical and molecular compositional changes of calcifications and their surrounding soft tissue in DCIS. Methods: Consecutive tissue section from 422 patients were analyzed using mid-infrared and Raman hyperspectral imaging and X-ray diffraction (XRD) and included (i) ‘DCIS controls’ (DCIS without recurrence) (n=193), (ii) ‘DCIS cases’ (DCIS which subsequently recurred as an invasive disease) (n=123), (iii) ‘synchronous DCIS with invasive cancer’ (n=44) and ‘benign’ (n=62) groups. Both parametrized and data mining approaches including cluster analysis, unsupervised and supervised multivariate statistical analysis and biomodelling based on leave-one-sample-out cross-validation were used to classify the data and develop DCIS prediction models. Results: Prediction modelling of 314 calcification and soft tissue spectral images from 170 patients (‘DCIS controls’=118 versus ‘DCIS cases’=52) showed an AUROC mean value of 0.85 in distinguishing DCIS that did not recur from DCIS that recurred as IBC. Prediction modelling of XRD data from 124 patients (‘DCIS controls’=67 versus ‘DCIS cases’=57) showed an AUROC mean value of 0.80. The calcification spectral features showed pathology specific changes in phosphate to carbonate ratio as well as changes in magnesium whitlockite content. The epithelial features showed changes in protein secondary structure and content indicating structural remodeling between the two groups. XRD parametrized data analysis corroborated these observations in terms of difference in whitlockite, as well as crystallinity/unit axis of the measured calcifications. The prediction models are currently being tested independently on the remaining sample data. Conclusion: Complementary chemical and molecular analysis of breast calcifications and soft tissue using spectroscopy and XRD imaging predicts the likely progression of DCIS to IBC. Subject to the ongoing validation of biomarkers, these approaches appear as promising DCIS risk assessment tools that can inform diagnosis, prognosis and treatment options. Citation Format: Jayakrupakar Nallala, Doriana Calabrese, Sarah Gosling, Esther Lips, Rachel Factor, Allison Hall, Sarah Pinder, Ihssane Bouybayoune, Elinor Sawyer, Lorraine King, Jeffrey Marks, Thomas Lynch, Donna Pinto, Jelle Wesseling, Shelley Hwang, Keith Rogers, Nick Stone, Grand Challenge PRECISION consortium. Molecular imaging of calcification and soft tissue defines prognosis of DCIS to invasive breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5561.

Study of the Molecular Structure of Proteins in Eggs under Different Storage Conditions

The aim of this study is to reveal the relationship between changes in the physical and chemical characterization of eggs during storage and to provide a new way to assess egg freshness at the molecular chemical level. In this study, the Fourier transform infrared (FTIR) spectra of egg albumin from 60 fresh eggs (ISA Brown layer) stored for 1, 10, 20, 30, and 40 days at 4°C and 10°C were measured ( n = 6 ), and the amide I band of the egg albumin spectra was quantified by Fourier deconvolution and curve fitting to obtain the composition of the protein molecular structure. The results showed that different storage temperatures and storage times led to protein denaturation, resulting in changes in the conformation of the protein secondary structure. With increasing storage temperature and storage time, the content of the ordered structures β-sheet and α-helix in the egg protein secondary structure decreased significantly ( p &lt; 0.05 ), and the content of the disordered structure β-turn and random coil increased significantly ( p &lt; 0.05 ). Based on the pattern of protein secondary structure changes during storage, it was found that fresh eggs were better stored at 4°C for up to 15 days. Spearman’s correlation analysis of egg protein height, Haugh unit, egg weight, and protein secondary structure in eggs stored at 4°C showed a significant positive correlation between β-sheet and α-helix and egg weight, protein height, and Haugh unit. And a significant negative correlation between random coil and egg weight, protein height, and Haugh unit. Therefore, a curve fitting method based on Fourier transform infrared spectroscopy of deconvoluted spectra is an effective method for evaluating egg quality and freshness.

Preparation of quinoa protein with ultrasound pretreatment and its effects on the physicochemical properties, structural and digestion characterizations

This study aimed to investigate the effects of ultrasound pretreatment on the yield and the physicochemical properties, structural and digestion characterizations of quinoa protein (QP). Results showed that under the conditions of ultrasonic power density of 0.64 W/mL, ultrasonication time of 33 min, and the liquid-solid ratio of 24 mL/g, the highest yield of QP at 68.403 % was obtained, which was significantly higher than that without ultrasound pretreatment at 51.26 ± 1.76 % (P < 0.05). Ultrasound pretreatment decreased the average particle size and ζ-potential but increased the hydrophobicity of QP (P < 0.05). However, no significant protein degradation and secondary structure changes of QP by ultrasound pretreatment were observed. In addition, ultrasound pretreatment slightly improved the in vitro digestibility of QP and reduced the dipeptidyl peptidase IV (DPP-IV) inhibitory activity of the hydrolysate of QP by in vitro digestion. Overall, this work demonstrates that ultrasound-assisted extraction is appropriate for improving the extraction efficiency of QP.

Abstract P4-02-22: Breast microcalcification chemistry predicts DCIS prognosis

Abstract Introduction: Microcalcifications are a common feature in mammographic detection of ductal carcinoma in situ (DCIS), and occur in &amp;gt;80% of cases. Known to be present as type I (calcium oxalate-CaO) and type II (carbonated calcium hydroxyapatite-CHAP) microcalcifications, their association with DCIS and their role in the progression of DCIS to invasive breast cancer (IBC) remains unexplored. In an effort to understand the factors involved in DCIS prognosis, it is hypothesized that changes in the chemical composition of calcifications, in tandem with molecular changes in the surrounding soft tissue, will define patients with DCIS who will progress to develop IBC from those who remain with a stable DCIS phenotype. To this end, a novel label-free approach of hyperspectral imaging using mid-infrared (mid-IR) and Raman spectroscopy was used to probe calcification chemistry and molecular composition of the surrounding ductal and stromal soft tissue. The main aim of the work is to identify biomarkers for DCIS prognosis, based on chemical and molecular compositional changes of calcifications and the surrounding soft tissue. It is anticipated that the spectral biomarkers will provide patients and clinicians an informed risk assessment whether to undertake treatment for DCIS or to be placed under active surveillance. Methods: Tissue samples from 422 patient have been obtained and include (i) ‘pure DCIS’ (DCIS without recurrence) (n=193), (ii) ‘DCIS with invasive recurrence’ (DCIS from patients who subsequently were known to develop invasive disease) (n=123), (iii) ‘DCIS plus contemporaneous invasive cancer’ (n=44) and ‘benign’ (n=62) samples. Serial tissue sections were measured using mid-IR and Raman hyperspectral imaging approaches targeting the same calcification and soft tissue regions from specific DCIS ducts. Hyperspectral imaging data was initially pre-processed to digitally remove paraffin and unintended spectral interferences. The pre-processed data was subjected to cluster analysis followed by unsupervised and supervised machine learning classification models to identify spectral features associated with DCIS and its progression to IBC. Results: Cluster analysis based segmentation of hyperspectral images revealed histopathological features including calcifications, epithelium, necrotic areas, connective tissue and stroma. Spectra were extracted from each of the histopathological features using image coordinates, and biomodelling analysis was performed. Initial analysis of 314 calcification images from 170 patients with (i) ‘pure DCIS’ (n=118) and (ii) ‘DCIS with invasive recurrence’ (n=52) showed an area under the receiver operating characteristic (AUROC) mean value of 85% in distinguishing pure DCIS from DCIS that later recurred as IBC. The calcification features appeared to indicate pathology specific changes in phosphate and carbonate content as well as changes in magnesium whitlockite content. Similar analysis of the surrounding soft tissue spectral features showed an AUROC mean value of 85% (necrotic regions surrounding calcifications) and 76% (epithelium) respectively. The epithelial features showed changes in protein secondary structure and content, which together with the calcification changes indicate structural remodelling in DCIS that progresses to IBC, from those that do not. Perspectives: In the ongoing analyses of imaging data from 422 patients, it is anticipated that molecular/structural features from calcification and soft tissue imaging data will provide important cues in understanding DCIS prognosis and could be a promising way forward in determining management of DCIS risk and treatment underpinned by the identification of specific discriminatory spectral markers. Acknowledgments: This work was supported by Cancer Research UK and by KWF Kankerbestrijding (ref. C38317/A24043). Citation Format: Jayakrupakar Nallala, Doriana Calabrese, Sarah Gosling, Esther Lips, Rachel Factor, Allison Hall, Sarah E. Pinder, Ihssane Bouybayoune, Lorraine King, Jeffrey Marks, Thomas Lynch, Donna Pinto, Jelle Wesseling, E Shelley Hwang, Keith Rogers, Nick Stone. Breast microcalcification chemistry predicts DCIS prognosis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-22.

Insight in changes in starch and proteins molecular structure of non-wheat cereal flours influenced by roasting and extrusion treatments

Barley, rye, triticale, oat, sorghum and millet flours, subjected to roasting or extrusion treatment, were analyzed by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) and Raman spectroscopy with aim to determine structural changes within starch and protein molecules that caused specific rheological properties of heat treated flours. Mixolab was used to determine how changes in starch and protein structure are reflected in rheological properties of dough. Extrusion treatment caused more extensive changes both in starch and protein structure than roasting treatment. Changes of protein secondary structure from α-helix to β-turn aggregated by hydrogen bonds, formed mainly between tyrosyl groups, as well as change in disulphide bond conformation, were more pronounced in extruded flour samples. Changes in the starch structure were also more extensive in extruded flours samples, including complete gelatinization and formation of amylose-lipid complexes. The results obtained by Mixolab showed that all extruded doughs showed resistance at C2 point, which was shifted to approximately 30 min, indicating higher mechanical resistance. It was supposed that this change in dough behaviour is related to protein polymerization during extrusion treatment and starch aggregation during roasting treatment.

Spectroscopic Studies of Quinobenzothiazine Derivative in Terms of the In Vitro Interaction with Selected Human Plasma Proteins: Part 2.

Synthesis of anticancer substances and studying their binding abilities towards human serum proteins as carriers are important parts of pharmaceutical and medical sciences development. The presented work is a continuation of studies of quinobenzothiazine derivatives binding with serum proteins. The main aim of this work was a spectroscopic analysis of second from benzothiazinium derivatives salt, 9-fluoro-5-alkyl-12(H)-quino [3,4-b][1,4]benzothiazinium chloride (Salt2), its interaction with carrier proteins, i.e., human serum albumin (HSA), α1-acid glycoprotein (AGP), human gamma globulin (HGG), and the study of protein secondary and tertiary structure changes using spectroscopic techniques (spectrofluorescence, UV-Vis and circular dichroism CD spectroscopy). In order to mimic in vivo conditions, control normal serum (CNS) was used. Using the Klotz method, both binding constants (Ka [M-1]) and the number of binding classes (n) were calculated. In addition, the percentage of displacement of binding site markers from HSA and AGP molecules has been defined. Based on the obtained data, it can be concluded that the main binding protein for Salt2 is AGP. HSA and HGG are also involved in the distribution of the studied substance in the bloodstream. Moreover, Salt2 very slightly interacts with CNS, which can cause strong therapeutic as well as toxic effects. The analysis of CD spectra confirms that there are no changes in the secondary structure of the main binding proteins in the presence of Salt2.

Role of NS2 specific RNA binding and phosphorylation in liquid-liquid phase separation and virus assembly.

Liquid-liquid phase separation (LLPS) has assumed a prominent role in biological cell systems, where it underpins the formation of subcellular compartments necessary for cell function. We investigated the underlying mechanism of LLPS in virus infected cells, where virus inclusion bodies are formed by an RNA-binding phosphoprotein (NS2) of Bluetongue virus to serve as sites for subviral particle assembly and virus maturation. We show that NS2 undergoes LLPS that is dependent on protein phosphorylation and RNA-binding and that LLPS occurrence is accompanied by a change in protein secondary structure. Site-directed mutagenesis identified two critical arginine residues in NS2 responsible for specific RNA binding and thus for NS2-RNA complex driven LLPS. Reverse genetics identified the same residues as essential for VIB assembly in infected cells and virus viability. Our findings suggest that a specific arginine-RNA interaction in the context of a phosphorylated state drives LLPS in this, and possibly other, virus infections.

Effect of microwave treatment on the structural and functional properties of proteins in lentil flour

Microwave is the commonly adopted thermal technology for the cooking and other industrial processing of lentils. The study focused on evaluating the effect of microwave treatment (MT) on the protein profile and functional properties of lentil flour (LF). The lentils with 18% moisture content were exposed to MT at three different power levels (540, 720, and 900 W) and exposure times (2, 4, and 6 min). The treated lentil flour samples were defatted before further studies. The results revealed that the MT positively impacted most of the desired functional properties like water and oil absorption capacity and emulsion properties; also, MT has an insignificant effect on foaming capacity, and significantly reduced the foaming stability from 80.25% to 60%. The structure of the MT flours differed from the untreated counterparts, and MT improved the functional properties of lentil flour. Protein secondary structural changes were observed in MT flour. It was observed that MT altered the crystalline and amorphous natures of the LF, implying that MT is appealing as an alternative emerging technology to improve the functional and quality parameters of lentil proteins with potential applications for other pulse proteins.

Potential of air-currents assisted particle separation (ACAPS) technology for hybrid fractionation of clean-label faba bean (Vicia faba L.) protein

Air-currents assisted particle separation (ACAPS) of faba beans prior to wet separation was evaluated for its impact on chemical composition, yield and recovery of protein, starch and dietary fibre, and protein secondary structural changes. The protein and dietary fibre contents of faba bean cultivars Snowbird and Athena were positively correlated (r = 0.9, P < 0.0001) with the increasing particle size, which was offset by starch contents. ACAPS treatment of faba bean dehulled bean flours resulted in both protein- and dietary fibre-rich coarse fractions (75–500 μm). Subjecting the ACAPS-treated protein-rich (Snowbird: 38.96 ± 0.33% and Athena: 37.54 ± 0.20%) coarse fractions (250–500 μm) to micellization resulted in protein isolates (Snowbird: 97.74 ± 0.18% and Athena: 94.30 ± 0.29%). This method also resulted in starch isolates (Snowbird: 94.05 ± 1.67% and Athena: 94.33 ± 0.53%) and dietary fibre concentrates (Snowbird: 54.41 ± 1.87% and Athena: 44.89 ± 1.40%) as co-products, which were “clean label” ingredients. FTIR data confirmed that hybrid fractionation had minimal impact on protein secondary structures with β-sheets as the major structures.

Studies on the interaction between homological proteins and anthocyanins from purple sweet potato (PSP): Structural characterization, binding mechanism and stability

The protein-bound anthocyanin complexes are naturally existed in food systems by their spontaneous interaction. In this study, the interaction mechanism of homological proteins (p-PSP) and anthocyanins (FAC-PSP) was investigated to explore the binding characteristic of native protein-bound anthocyanins from purple sweet potato (p-BAC-PSP). The structural characterization, stability and anti-ultraviolet property of p-BAC-PSP were also evaluated. Results revealed that hydrophobic interaction is dominant binding force for forming p-BAC-PSP. The binding resulted in protein secondary structure changes with more β-sheet and lower β-turn, random coil structures. Fluorescence spectroscopy demonstrated that FAC-PSP quenched p-PSP fluorescence in a combination of static and dynamic mode (static dominant) with a binding constant of 105 L/mol reflecting strong affinity of FAC-PSP to p-PSP. Moreover, the complex form exhibited better protective effects on anthocyanins for pH, light, thermal stabilities and higher anti-ultraviolet activity. These findings further expanded the application of anthocyanins as stable, functional food and cosmetic ingredients.

Phenotypic and genetic analyses of four cases of coagulation factor XII deficiency

ABSTRACT Objectives To identify the clinical phenotypic and molecular pathogeneses of four cases of coagulation factor XII deficiency and to deepen the cognition of this disease. Methods Coagulation tests were performed through one stage of coagulation on a STAGO coagulation analyser. Coagulation factor XII antigen was detected using enzyme-linked immunosorbent assay. The species conservatism and structural change of mutant proteins were analysed using MegAlign and PYMOL. Meanwhile, missense variants and a novel splice site variant were identified using PolyPhen2 and NetGene2. Results The four cases had an observably prolonged activated partial thromboplastin time but without obvious bleeding tendency. Their coagulation factor XII activity (FⅫ:C) and antigen (FXII:Ag) were greatly reduced. Six mutations were detected: NM_000505.4:c.398-1G>A, NP_000496.2:p.(Pro182Leu), NP_000496.2:p.(Ser479Ter), NP_000496.2:p.(Cys559Arg), NC_000005.10:g.7217_7221delinsGTCTA and NM_000505.4:c.1681-1G>A. The first five are newly discovered mutations. The two missense mutation sites were highly conservative, and their protein secondary structure changes may occur not only on the mutation sites but also on other domains. In silico analysis revealed that NP_000496.2:p.(Pro182Leu) may be BENIGN, NP_000496.2:p.(Cys559Arg) may be damaging, and that NM_000505.4:c.398-1G>A and NM_000505.4:c.1681-1G>A are crucial for splicing. Conclusion We found six types of mutations, of which five were novel. The two missense mutation sites might be closely related to the function of coagulation factor XII. The mutations were the primary culprits of factor XII deficiency.

Microencapsulating role of whey protein isolate and sucrose in protecting the cell membrane and enhancing survival of probiotic lactobacilli strains during spray drying, storage, and simulated gastrointestinal passage

Limosilactobacillus reuteriKUB-AC5 andLactobacillus johnsoniiKUNN19-2 were spray-dried using whey protein isolate, sucrose, and whey protein isolate combined with sucrose at 130 °C and 150 °C. Differences in membrane fatty acid compositions of probiotics led to differences in cell membrane fluidity, cell injury, and cell survival during spray drying. Combination of whey protein isolate and sucrose improved survival of L. reuteriKUB-AC5, whose cell membrane were more fluid and more sensitive to stress, while whey protein isolate provided the best protection for L. johnsoniiKUNN19-2 against changes in protein secondary structures. Moreover, a relationship was found between membrane damage and the shelf-life ofmicrocapsules. Both microencapsulated cells prepared at the inlet temperature of 130 °C showed an enhanced survival upon simulated gastric digestion compared to the free cells. A thorough understanding of injury sites during spray drying will contribute to establishing optimal conditions and encapsulant formulations that maintain the cells’ viability during spray drying, storage, and gastrointestinal passage.