Intrinsic Protein Fluorescence Research Articles

Enhancement of vitamin B stability with the protection of whey protein and their interaction mechanisms

Vitamin B is easily degraded by light and heat during storage, which results in nutritional loss of food. Whey protein is expected to protect vitamin B by forming complexes through secondary bonds. The properties of the complexes and protective effects of whey protein on vitamins B1, B2, B3 and B6 were characterized. The percentage losses of vitamin B were decreased by more than 60% with the protection of whey protein. FTIR, fluorescence spectroscopy, thermodynamic analysis and molecular docking were used to investigate the binding interaction between vitamin B and whey protein. Vitamin B quenched the intrinsic fluorescence of whey protein, mainly with a static nature (Kq > 2.0 × 1010 L/(mol·s)). The interactions between whey protein and vitamin B were mostly mediated by hydrogen bonds and van der Waals forces, as demonstrated by the thermodynamic parameters and molecular docking.

New Ru(II)-p-cymene compounds bearing indomethacin and indomethacin-pyridineamide ligands: synthesis, characterization, computational studies and investigation of their interactions with the Human Serum Albumin

The research interest in ruthenium(II) organometallic compounds has continually increased due to their promising performance in biological assays targeting diverse medicinal applications. This work reports the interactions of the [Ru(II)-η6-p-cymene] framework with the indomethacin (HL1) drug and with its modified analogue indomethacin-pyridineamide (L2). Two new half-sandwich organometallics, [Ru(η6-p-cymene)(L1)Cl] (1) and [Ru(η6-p-cymene)(L2)Cl2] (2), were successfully synthesized and fully characterized by ESI-MS, NMR, ATR-FTIR and UV/VIS spectroscopy. The chemical and electronic structures were corroborated by DFT computational calculations. Compound 2 was chemically stable, keeping the L2 ligand in the coordination sphere in coordinating solvents, and was investigated for the interaction with the Human Serum Albumin (HSA) protein by fluorescence quenching studies. This compound acted as a quencher to the intrinsic protein fluorescence, mainly through binding-related quenching (static) mechanism, showing high value for the apparent association constant (Ka). The analysis of the thermodynamic parameters revealed spontaneous process, presenting low temperature dependence, with electrostatic forces playing a key role in the HSA-compound 2 adduct binding. Site competitive studies suggested that the organometallic 2 may compete for the binding site of ibuprofen (subdomain IIIA) and also for the binding site of Ru(III) naked ion (subdomain IB or IIA). Molecular docking simulations showed that both Ru(II)-arenes may interact through hydrogen bonding with arginine residues, and exhibit high affinity for the microenvironment located in subdomains IIIA and IB. These findings suggest that the HSA protein might be a potential carrier for these Ru(II)-organometallic compounds in the blood plasma.

Inhibitory activity of luteolin on α‐glucosidase and pancreatic lipase in vitro and in vivo

SummaryObesity is becoming a top global health problem. Inhibition of digestive enzymes is an effective approach in weight management due to the reduction of energy absorption. In this study, the inhibitory effects of luteolin on α‐glucosidase and pancreatic lipase were investigated in vitro and in vivo via enzyme kinetics, fluorescence quenching, molecular docking and rat experiments. The results showed that luteolin inhibited α‐glucosidase and pancreatic lipase in a mixed‐type and uncompetitive manner with IC50 values of 29.31 and 42.74 μg mL−1 respectively. The binding of luteolin with the two enzymes caused the quenching of intrinsic fluorescence of protein. Molecular docking simulation revealed that luteolin could be located in the active pocket of α‐glucosidase and interact with the key amino acid residues. Moreover, a hydrophobic interaction and a van der Waals force were formed between luteolin and the key amino acid residues of pancreatic lipase (His264 and Ser153). In vivo studies further confirmed that luteolin could significantly decrease the absorption of glucose and triacylglyceride in rats by inhibiting α‐glucosidase and pancreatic lipase.

Insights on the in-vitro binding characteristics of human α-1-acid glycoprotein (HAG) with JAK inhibitor ocalcitinib: Multi-spectroscopic analysis combined with theoretical calculations

A powerful Janus kinase (JAK) inhibitor, ocalcitinib has been authorized for controlling pruritus and atopic dermatitis from canine allergic dermatitis and many kinds of cytokines. To further understand the drug action, the interaction between ocalcitinib and HAG was explored using multi-spectroscopic approaches combined with theoretical calculations. The results demonstrated that ocalcitinib effectively quenched the intrinsic fluorescence of HAG protein through a mixed quenching and formed a stable ocalcitinib–HAG complex that had the association constant of 104–105M−1, outlining that ocalcitinib binding to HAG protein had a moderate affinity. In the ocalcitinib–HAG complex, ocalcitinib implanted into the bucket-shaped hydrophobic chamber of HAG, leading to the slight variety in the hydrophobicity of the microenvironment encircled Trp-and Tyr-resides as well as the secondary structure of HAG. And, the driving forces that forms the ocalcitinib-HAG complex were mainly the hydrogen bonding interaction, Van der Waals forces, and hydrophobic interaction. It is also confirmed that the common metal ions like Ca2+, Mg2+, Fe3+, Zn2+, Cu2+, Co2+, Ni2+ ions) except K+ ion had a greater negative influence on the complexation between ocalcitinib and HAG. This study should conducive to estimating the pharmacological natures of ocalcitinib, comprehending the distribution and operation of the drug in the body, and developing novel medication devise.

Effect of temperature and pH on the structure and stability of tumor-specific lectin jacalin and insights into the location of its tryptophan residues: CD, DSC and fluorescence studies

Jacalin, the jackfruit seed lectin, exhibits high specificity for the tumor-specific T-antigen and is used in various biomedical and biotechnological applications. Here, we report biophysical studies on the thermal unfolding of jacalin and the effect of pH and temperature on its secondary structure. Differential scanning calorimetric (DSC) studies revealed that native jacalin unfolds at ∼60 °C and that carbohydrate binding stabilizes the protein structure. Circular dichroism spectroscopic studies indicated that the secondary structure of jacalin remains mostly unaffected over pH 2.0–9.0, whereas considerable changes were observed in the tertiary structure. DSC experiments demonstrated that jacalin exhibits two overlapping transitions between pH 2 and 5, which could be attributed to dissociation of the tetrameric protein into subunits and their unfolding. Interestingly, only one transition between pH 6 and 9 was observed, suggesting that the subunit dissociation and unfolding occur simultaneously. While quenching of the protein intrinsic fluorescence by acrylamide increased significantly upon carbohydrate binding, quenching by succinimide is essentially unaffected. We attribute this difference to increased exposure of Trp-123 in the α-chain as it is involved in carbohydrate binding. Both acrylamide and succinimide gave biphasic Stern-Volmer plots, consistent with differential accessibility of the two tryptophan residues of jacalin to them.

Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03–10 μg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.

Spectroscopic and thermodynamic characterization of the interaction between sugar-stabilised silver nanoparticles and wheat germ agglutinin (WGA), a chitin binding lectin

Nanomaterials have lately been investigated in agriculture as eco-friendly and effective antifungal agents. Many nanomaterials, notably metal nanoparticles, have strong antifungal properties. Among metal nanoparticles, Ag nanoparticles have received the most attention as antifungal agents. Many plant lectins have been identified as antifungal agents. Conjugating AgNPs with antifungal lectins is thus expected to improve Ag nanoparticle antifungal efficacy. Understanding the molecular interactions and physical features of lectin-sugar-stabilised nanoparticle conjugates is critical for future applications. WGA has traditionally been used as an anti-tumor and antifungal agent. To investigate the prospect of developing an effective biocompatible antifungal system with applications in medicine and agriculture, fluorescence spectroscopy was used to investigate the interaction between sugar-stabilised silver nanoparticles and WGA. During the association, protein intrinsic fluorescence emission is suppressed by about ∼15 % at saturation, with no significant shift in fluorescence emission maxima. Binding tests reveal a strong bond. Stern-Volmer analysis of the quenching data indicates that the interaction happens via a static quenching process that induces complex formation. The study of hemagglutination activity and interaction experiments in the presence of particular sugar shows that the lectin's sugar-binding site is separate from the nanoparticle-binding site, and cell recognition is conserved in the lectin-nanoparticle complex. The Van't Hoff plot thermodynamic parameters suggest that the contact is hydrophobic. The fact that ΔGo is negative shows that the binding is a spontaneous process. CD spectroscopy experiments reveal that the lectin's secondary structure is not affected while binding to the nanoparticle. Our findings suggest that a stable WGA-silver nanoparticle combination may emerge for a variety of applications.

Dark nanodiscs for evaluating membrane protein thermostability by differential scanning fluorimetry

Measuring protein thermostability provides valuable information on the biophysical rules that govern the structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently developed nonfluorescent membrane scaffold protein to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the “dark” nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs loaded with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) lipids show a complex biphasic thermal unfolding pattern with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (Tm) of 70.5°C and 77.5°C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a DMPC and DMPG nanodisc. To extend the utility of this method, we evaluate and compare the thermostability of DsbB in different lipid environments. We introduce this method as a new tool that can be used to understand how compositionally and biophysically complex lipid environments drive membrane protein stability.

Purification, Characterization, and Ferrous Oxidation Kinetics of Iron Oxidase from Acidithiobacillus ferridurans

Iron oxidase was proposed to be the initial electron acceptor from the ferrous ion in the iron oxidation of the Acidithiobacillus genus for metal bioextraction; however, its most fundamental property of direct ferrous oxidation kinetics remains undetermined due to the confusion of reaction monitor method. Here, a recombinant iron oxidase from Acidithiobacillus ferridurans Riv11 was constructed, expressed, purified, characterized, and further used to investigate the kinetics and mechanism of ferrous oxidation. This protein is more stable in an acid solution than in a neutral solution. An infrared characteristic peak around 1050 cm−1 of the [Fe4S4] cluster was identified. The [Fe4S4] cluster does not affect the secondary structure of protein, but plays an important role in the stability of protein and strongly absorbs the intrinsic fluorescence of protein, resulting in a great loss of the fluorescence emission. The protein has far more absorbance than those of the iron ions in solution in the visible region; therefore, the maximum difference absorbance around 500 nm between the oxidized and reduced states of protein can be used to monitor the reaction of ferrous oxidation. Accordingly, the kinetic rate constant of the reaction was determined. Bioinformatics analysis and molecular simulation further revealed the underlying molecular mechanism that ferrous ions approach the protein at the edge of a large hydrophobic surface patch nearest to the [Fe4S4] cluster from a direction far from all positively charged residues, which enough enables an efficient electron transfer.

2,2′-Bithiophene as sensor tag for ligand–protein binding assays based on Förster resonance energy transfer

Ligand–protein binding assays based on intrinsic protein fluorescence are straightforward, inexpensive methods to study ligand–protein interactions. However, their applicability is limited to ligands that can interfere with protein emission. In this Note, we describe the applicability of 2,2′-bithiophene as a FRET-based sensor tag, that can be incorporated into high-affinity ligands to generate target-specific compounds able to quench protein fluorescence upon binding. The generated ligands were assessed in different assay designs. Considerations to account for possible sources of interference with the assay readout are addressed, besides interpretation of the obtained results.

Effect of Capsaicin and Dihydrocapsaicin in Capsicum on Myofibrillar Protein in Duck Meat.

Spice and its extracts have gained widespread utilization as natural and eco-friendly additives, imparting enhancements in flavor, color, and antioxidative attributes to meat-based products. This work aims to study the effect mechanism of capsaicin (CA) and dihydrocapsaicin (DI) in capsicum (chili pepper) on the structure and function of myofibrillar proteins (MPs) in duck meat during thermal treatment. The results showed that at a CA-DI to MP ratio of 1:500 (g/g) following a 12 min heat treatment, the carbonyl content of MPs in duck meat decreased by 48.30%, and the sulfhydryl content increased by 53.42%. When the concentration was 1:500 (CA-DI, g/g) after 24 min of heat treatment, the •OH and DPPH radical scavenging rates were highest at 59.5% and 94.0%, respectively. And the initial denaturation temperature of MPs was the highest at 96.62 °C, and the thermal absorption was lowest at 200.24 J g-1. At the parameter, the smallest particle size and size distribution range of MP were 190 nm (9.51%). Furthermore, the interplay between CA-DI and MPs contributed to a reduction in the protein particle size and intrinsic fluorescence. In summary, the combination of CA-DI and MPs played a crucial role in inducing protein unfolding and disintegration.

Insights into Intramuscular Connective Tissue Associated with Wooden Breast Myopathy in Fast-Growing Broiler Chickens.

Wooden breast myopathy (WBM) is a meat abnormality affecting pectoralis majors (PMs) of fast-growing broiler chickens. WBM-affected PMs exhibited varied meat qualities with increasing WBM severity. Normal PMs (NOR), mild WBM-affected PMs (MIL), moderate WBM-affected PMs (MOD), and severe WBM-affected PMs (SEV) were selected as raw materials. The structure and organization of connective tissue and fibrillar collagen were investigated through immersing with sodium hydroxide solution, Masson trichrome staining, and using an electron microscope. The mechanical strength of intramuscular connective tissue was analyzed via the shear force of samples treated with sodium hydroxide solution. The thermal property and secondary structure of connective tissue were analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The obtained connective tissue was dissolved in a sodium hydroxide solution for the evaluation of the physicochemical properties of proteins, including particle size, molecular weight, surface hydrophobicity, and intrinsic fluorescence. In particular, the particle size was measured using a zeta potential instrument. The molecular weight was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The surface hydrophobicity and intrinsic fluorescence were measured by spectroscopy technology. Histologically, macrophage infiltration, myodegeneration and necrosis, regeneration, fibrous connective tissue, and thickened perimysial connective tissue were observed in WBM-affected PMs, especially SEV with fibrosis, including blood vessels. Compared with NOR, WBM led to increased average diameter of the collagen fibrils in perimysial (36.61 nm of NOR to 69.73 nm of SEV) and endomysial (34.19 nm of NOR to 56.93 nm of SEV) layers. A significant increase (p < 0.05) was observed in the mechanical strength (2.05 N to 5.55 N) of fresh PMs and the thermal transition temperature (onset temperature (TO), 61.53 °C to 67.50 °C; maximum transition temperature (TM), 66.46 °C to 70.18 °C; termination temperature (TE), 77.20 °C to 80.88 °C) of connective tissue from NOR to SEV. Cooking decreased the mechanical strength, and MOD samples showed the highest mechanical strength (1.24 N, p < 0.05), followed by SEV (0.96 N), MIL (0.93 N), and NOR (0.72 N). For proteins in connective tissue, random coil (19.64% to 29.61%, p < 0.0001), particle size (p < 0.05), and surface hydrophobicity (p < 0.05) increased with the decrease in the α-helix (14.61% to 11.54%, p < 0.0001), β-sheet (45.71% to 32.80%, p < 0.0001), and intrinsic fluorescence of proteins from NOR to SEV. The molecular weights of intramuscular connective tissue proteins were in the ranges of >270 kDa, 180-270 kDa, 110-180 kDa, 95-100 kDa, and <15 kDa. Taken together, WBM resulted in thickened organization, tightly packed collagen fibrils, increased mechanical strength and thermal temperature, and increased particle size, surface hydrophobicity, and intrinsic fluorescence of proteins in connective tissue, as the WBM severity increased.

Cloning, expression and purification of the outer membrane protein N from Gram-negative bacterial strains

To employ polyclonal antibodies against 6his-tags to detect the expression of recombinant EcOmpN in the E. coli BL21 (DE3) Omp8 Rosetta strain using a Western blot, the ompN gene segments from four Gram-negative bacterial strains (including E. coli of BL21 (DE3) Omp8 Rosetta, DH5α, Origami, and XL1-blue) were combined with the pET21b(+) vector to create recombinant plasmids, known as pET21b(+)/ompN-BOR, pET21b(+)/ompN-DHA, pET21b(+)/ompN-Ori, and pET21b(+)/ompN-XLB for production of four recombinant EcOmpN proteins, called BL21 (DE3) Omp8 Rosetta (BOR), DH5α (DHA), Origami (Ori) and XL1-blue (XLB), respectively. Each recombinant plasmid-carrying bacterial strain was induced with 0.5 mM IPTG for 4 h. Crude recombinant EcOmpN proteins were separated on a 12% SDS-PAGE and transferred to a nitrocellulose membrane and treated separately with anti-OmpN and anti-histidine tag polyclonal antibodies. E. coli BL21 (DE3) Omp8 Rosetta strains fully produced all four recombinant EcOmpN proteins as inclusion bodies. Following protein refolding procedures, the secondary structure of recombinant BOR protein was likewise shown in the stable trimeric form, whereas recombinant DHA, XLB and Ori proteins were demonstrated in monomeric form. The intrinsic fluorescence and substance interaction of recombinant BOR protein were further established and showed that this protein reacted with kanamycin more intensely than ampicillin.

Immobilized biocatalyst engineering: Biocatalytic tool to obtain attractive enzymes for industry

Biocatalysis can improve current bioprocesses by identifying or improving enzymes that withstand harsh and unnatural operating conditions. Immobilized Biocatalyst Engineering (IBE) is a novel strategy integrating protein engineering and enzyme immobilization as a single workflow. Using IBE, it is possible to obtain immobilized biocatalysts whose soluble performance would not be selected. In this work, Bacillus subtilis lipase A (BSLA) variants obtained through IBE were characterized as soluble and immobilized biocatalysts, and how the interactions with the support affect their structure and catalytic performance were analyzed using intrinsic protein fluorescence. Variant P5G3 (Asn89Asp, Gln121Arg) showed a 2.6-fold increased residual activity after incubation at 76 °C compared to immobilized wild-type (wt) BSLA. On the other hand, variant P6C2 (Val149Ile) showed 4.4 times higher activity after incubation in 75 % isopropyl alcohol (36 °C) compared to Wt_BSLA. Furthermore, we studied the advancement of the IBE platform by performing synthesis and immobilizing the BSLA variants using a cell-free protein synthesis (CFPS) approach. The observed differences in immobilization performance, high temperature, and solvent resistance between the in vivo-produced variants and Wt_BSLA were confirmed for the in vitro synthesized enzymes. These results open the door for designing strategies integrating IBE and CFPS to generate and screen improved immobilized enzymes from genetic diversity libraries. Furthermore, it was confirmed that IBE is a platform that can be used to obtain improved biocatalysts, especially those with an unremarkable performance as soluble biocatalysts, which wouldn't be selected for immobilization and further development for specific applications.

Cannabidiol alleviates neuroinflammation and attenuates neuropathic pain via targeting FKBP5

Microglia is a heterogeneous population that mediates neuroinflammation in the central nervous system (CNS) and plays a crucial role in developing neuropathic pain. FKBP5 facilitates the assembly of the IκB kinase (IKK) complex for the activation of NF-κB, which arises as a novel target for treating neuropathic pain. In this study, cannabidiol (CBD), a main active component of Cannabis, was identified as an antagonist of FKBP5. In vitro protein intrinsic fluorescence titration showed that CBD directly bound to FKBP5. Cellular thermal shift assay (CETSA) indicated that CBD binding increased the FKBP5 stability, which implies that FKBP5 is the endogenous target of CBD. CBD was found to inhibit the assembly of the IKK complex and the activation of NF-κB, therefore blocking LPS-induced NF-κB downstream pro-inflammatory factors NO, IL-1β, IL-6 and TNF-α. Stern-Volmer analysis and protein thermal shift assay revealed that tyrosine 113 (Y113) of FKBP5 was critical for FKBP5 interacting with CBD, which is consistent with in silico molecular docking simulation. FKBP5 Y113 mutation (Y113A) alleviated the effect of CBD inhibiting LPS-induced pro-inflammatory factors overproduction. Furthermore, systemic administration of CBD inhibited chronic constriction injury (CCI)-induced microglia activation and FKBP5 overexpression in lumbar spinal cord dorsal horn. These data imply that FKBP5 is an endogenous target of CBD.

Interactions of Different Urolithins With Bovine Serum Albumin

Backgound/Objectives Urolithins (UROs) are the metabolites derived from the gut microbial action on ellagitannins and ellagic acid-rich foods. Following their absorption in the intestine, UROs are transported through the systemic circulation to various tissues where they can express their biological function as antimicrobial, anti-inflammatory, and anticancer agents. In addition to blood plasma, where they can be found as glucuronide and sulfate conjugates, they are also found in urine. Therefore, the interactions of UROs with serum proteins are of great clinical interest. Methods A powerful technique for examining these urolithin-serum protein interactions is fluorescence spectroscopy. Bovine serum albumin (BSA) is a particularly suitable model protein because it is readily available, affordable, and similar to human serum albumin. This work aimed to study the binding of UROs (urolithin A, UROA and urolithin B, UROB) and their glucuronide conjugates (UROAG and UROBG) to BSA by quenching the intrinsic fluorescence of protein. Results The spectra obtained showed that the binding process is influenced by the polyphenol's structure and the conjugation process with the glucuronide. The calculated Stern Vollmer binding constants (Ksv): UROA and UROB Ksv were 59236 ± 5706 and 69653 ± 14922, respectively, while for UROAG and UROBG, these values were 15179 ± 2770 and 9462 ± 1955, respectively, which showed that the binding affinity decreased with glucuronidation. Molecular docking studies confirmed that all of the studied molecules will bind favorably to BSA. The preferential binding site for both UROs and UROGs is Sudlow I, while UROs will also bind to Sudlow II. URO-Gs can bind to BSA in the cleft region with lower binding scores than for the Sudlow I binding site. Conclusion The aglycone's higher hydrophobicity increases the binding affinity to BSA, thus reducing its bioavailability in the blood.

Exploration of molecular interaction between different plant proteins and 2-pentylfuran: Based on multiple spectroscopy and molecular docking.

Soy protein, peanut protein, and wheat protein are commonly applied in plant-based products, but specific off-odor makes it difficult for consumers to accept, with 2-pentylfuran being one of the most representative flavors. In this study, 2-pentylfuran was employed as an example to explore the behavior and mechanism of the three proteins in absorbing off-odors. Gas chromatography-mass spectrometry (GC-MS) analysis indicated that different plant proteins were both able to adsorb 2-pentylfuran. Circular dichroism proved 2-pentylfuran could drive the α-helix to β-sheet transition of SPI, which was not obvious in PnPI and WPI. Ultraviolet spectroscopy tentatively determined that 2-pentylfuran caused changes in the tyrosine and tryptophan microenvironments of different plant proteins, which were further evidenced by synchronous fluorescence at fixed wavelength intervals of 15 nm and 60 nm. Static quenching of protein intrinsic fluorescence indicated that they formed a stable complex with 2-pentylfuran, except for WPI (dynamic quenching). The various conformations of the three proteins are the main reason for the difference in flavor retention of protein. Soy protein, peanut protein, and wheat protein adsorbing 2-pentylfuran rely on non-covalent forces, especially hydrophobic interactions, maintained between the protein and 2-pentylfuran. This article is protected by copyright. All rights reserved.

Primary structure determination and physicochemical characterization of DSP-3, a phosphatidylcholine binding glycoprotein of donkey seminal plasma

Major proteins of the seminal plasma in a variety of mammals such as bovine PDC-109, equine HSP-1/2, and donkey DSP-1 contain fibronectin type-II (FnII) domains and are referred to as FnII family proteins. To further our understanding on these proteins, we carried out detailed studies on DSP-3, another FnII protein of donkey seminal plasma. High-resolution mass-spectrometric studies revealed that DSP-3 contains 106 amino acid residues and is heterogeneously glycosylated with multiple acetylations on the glycans. Interestingly, high homology was observed between DSP-1 and HSP-1 (118 identical residues) than between DSP-1 and DSP-3 (72 identical residues). Circular dichroism (CD) spectroscopic and differential scanning calorimetric (DSC) studies showed that DSP-3 unfolds at ~45 °C and binding of phosphorylcholine (PrC) – the head group moiety of choline phospholipids – increases the thermal stability. Analysis of DSC data suggested that unlike PDC-109 and DSP-1, which exist as mixtures of polydisperse oligomers, DSP-3 most likely exists as a monomer. Ligand binding studies monitoring changes in protein intrinsic fluorescence indicated that DSP-3 binds lyso-phosphatidylcholine (Ka = 1.08 × 105 M−1) with ~80-fold higher affinity than PrC (Ka = 1.39 × 103 M−1). Binding of DSP-3 to erythrocytes leads to membrane perturbation, suggesting that its binding to sperm plasma membrane could be physiologically significant.

Probing the chemical unfolding and phospholipid binding to the major protein of donkey seminal plasma, DSP-1 by fluorescence spectroscopy

Fibronectin type-II (FnII) proteins which constitute the major fraction in the seminal plasma of many mammals, bind to choline phospholipids on the spermatozoa and play a crucial role in sperm capacitation, which enables the sperm to fertilize the egg. In the current study, we have investigated the heterogeneity of microenvironment around the tryptophan residues in the major FnII protein of donkey seminal plasma, DSP-1, in the native state and upon ligand binding employing fluorescence methods. Steady-state and time-resolved fluorescence studies on the quenching of the protein intrinsic fluorescence employing neutral and ionic quenchers revealed that the environment of the tryptophan residues in DSP-1 is less heterogeneous as compared to other homologous seminal plasma FnII proteins PDC-109 (bovine) and HSP-1/2 (equine). The quenching was decreased by ligand binding with choline containing lipids, Lyso-PC and DMPC providing stronger shielding of the Trp residues than the soluble ligand, phosphorylcholine, which is most likely due to segment(s) of the protein penetrating into the hydrophobic interior of the lipid membrane/micelles. Studies on the chaotrope-induced unfolding of DSP-1 revealed that disulfide bonds in the FnII domains of the protein prevent its complete unfolding, and that cooperativity of unfolding is also modulated by polydispersity of the protein.

Interaction of sugar stabilised silver nanoparticles with Momordica charantia seed lectin, a type II ribosome inactivating protein.

Sugar-stabilised nanomaterials have received a lot of attention in cancer therapy in recent years due to their pronounced application as specific targeting agents and maximizing their therapeutic potential while bypassing off-target effects. Lectins, the carbohydrate-binding proteins, are capable of binding to receptors present on the target cell/tissue and interact with transformed glycans better than normal cells. Besides some of the lectins exhibit anticancer activity. Conjugating sugar-stabilised NPs with lectins there for is expected to multiply the potential for the early diagnosis of cancer cells and the specific release of drugs into the tumor site. Because of the prospective applications of lectin-sugar-stabilised nanoparticle conjugates, it is important to understand their molecular interaction and physicochemical properties. Momordica charantia Seed Lectin (MCL) is a type II RIP and has been known as an anti-tumor agent. Investigation of the interaction between sugar-stabilised silver nanoparticles and MCL has been performed by fluorescence spectroscopy to explore the possibility of creating an effective biocompatible drug delivery system against cancer cells. In this regard interaction between lectin and NPs should be well-preserved, while recognizing the specific cell surface sugar. Therefore experiments were carried out in the presence and absence of specific sugar galactose. Protein intrinsic fluorescence emission is quenched at ~ 20% at saturation during the interaction without any significant shift in fluorescence emission maximum. Binding experiments reveal a good affinity. Tetrameric MCL binds to a single nanoparticle. Stern-Volmer analysis of the quenching data suggests that the interaction is via static quenching leading to complex formation. Hemagglutination experiments together with interaction studies in the presence of specific sugar show that the sugar-binding site of the lectin is distinct from the nanoparticle-binding site and cell recognition is very much intact even after binding to AgNPs. Our results propose the possibility of developing MCL-silver nanoparticle conjugate with high stability and multiple properties in the diagnosis and treatment of cancer.