Polyphenol binding disassembles glycation-modified bovine serum albumin amyloid fibrils.

Glycation of protein results in the formation of advanced glycation end-products (AGEs) and leads to deposition as amyloid fibrils. Adhesive structural properties of polyphenols to aromatic amino acids draw significance in promoting, accelerating and/or stabilizing on-pathway and off-pathway folding intermediates, although the mechanistic action remains unclear. In this study, polyphenols remodeling mature AGEs modified amyloid fibrils were investigated through UV-visible spectroscopy, fluorescence spectroscopy, transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, MALDI-MS/MS analysis and molecular docking studies. Our findings confirmed the glycation-mediated transformation of native protein into ß-sheet rich amyloid fibrils. SDS-PAGE results suggested the presence of shorter peptide fragments ranging from ~10 kDa to ~40 kDa. MALDI-MS/MS results identified the plausible sequences to be His105-His181, Arg193-Lys242, Leu325-Tyr410, and Ala451-Tyr529. TEM and AFM results suggested that polyphenols binding mature amyloid fibrils remodel/disassemble them into distinct aggregate structures or non-amyloid fibrils. Circular dichroism studies suggested that polyphenols upon binding amyloid fibrils stabilizes and transforms the secondary structure towards helical or random coil-like conformation. Molecular modeling studies suggested high binding affinity and hydrophobic interaction to be the main driving force in remodeling perspective. Together, our findings suggest that polyphenols could differentially remodel mature AGEs-modified amyloid fibrils into distinct aggregate structures through non-covalent interactions and can alleviate AGEs-mediated amyloidosis.

Self-assembly of N-terminal Alzheimer's ß-amyloid and its inhibition.

Peptide sequence modulates amyloid fibril formation and triggers Alzheimer's disease. The N-terminal region of amyloid peptide is disordered and lack any specific secondary structure. An ionic interaction of Aß1-11 with factor XII is critical for the activation of the contact system in Alzheimer's disease. In this study, we report the self-assembly of fluctuating N-terminal Aß1-11 into nanotubes using atomic force micrography, transmission electron microscopy, circular dichroism studies and molecular modeling studies. The effect of four polyphenols: baicalein, rutin, vanillin and cyanidin-3-O-glucoside (C3G) was also explored on the amyloid fibril inhibitor perspective using amyloid specific dye Thioflavin T (ThT). AFM micrographs suggested the self-assembly of Aß1-11 into nanotubes after three weeks of incubation. Microwave treatment results in the conformational variation of disordered structure to ß-sheet rich amyloid fibrils. The presence of salts (sodium and potassium chloride) induces the structural transformation of Aß1-11 to super-helix. Fluorescence spectroscopy studies using ThT suggested differential inhibition of amyloid fibrils formation in the presence of polyphenols. Molecular modeling studies suggested that binding of polyphenols to Aß1-11 through hydrophobic interaction (Phe4 and Tyr 10) and hydrogen bonding (Glu3 and Arg5) play a substantial role in stabilizing Aß1-11-polyphenols complex. In the presence of polyphenols, Aß1-11 transforms to hybrid nanostructures thus hindering amyloid fibril formation. These results provide structural insights and importance of the N-terminal residues in the Aß1-42 self-assembly mechanism.

Polyphenols redirects the self-assembly of serum albumin into hybrid nanostructures.

Chronic hyperglycemia results in the formation of advanced glycation end-products (AGEs) and triggers amyloid fibril formation. Molecules designed to inhibit amyloid fibrils function by eliminating toxic oligomers or reducing fibril formation. Here, the bioactivity of polyphenols in redirecting the self-assembly of amyloid fibrils was reported through microscopic, spectroscopic and molecular docking studies. Our findings illustrate that glycation causes BSA to self-assemble into amyloid fibrils. 17 Lys residues had modified to carboxy methyl lysine (CML) but only Lys523 was probable of modifying into carboxy ethyl lysine (CEL). In contrast, only 6 Arg residues are identified to be modified to Argpyrimidine (Arg-p). A simple polyphenol baicalein (BLN) redirect the self-assembly of amyloid fibrils into off-pathway hybrid nanostructures. Circular dichroism spectroscopic studies suggested that in the presence of BLN helical conformation was favored. Molecular modeling studies suggested that hydrogen bonding and hydrophobic interaction of polyphenols preferentially at crucial amyloidogenic regions can hinder amyloid fibrillation (Phe133, Lys136, Tyr137, Ile141, Tyr160 and Arg185). Mass spectrometric results illustrated that the presence of a simple polyphenol BLN several residues are unmodified to CML, CEL or Arg-p. Together, our findings suggest that polyphenols could have a protective effect and the redirection can help alleviate the amyloid fibril formation.

Spectroscopic and molecular docking studies on the interaction of phycocyanobilin with peptide moieties of C-phycocyanin.

The binding of C-phycocyanin (CPC), a light harvesting pigment with phycocyanobilin (PCB), a chromophore is instrumental for the coloration and bioactivity. In this study, structure-mediated color changes of CPC from Spirulina platensis during various enzymatic hydrolysis was investigated based on UV-visible, circular dichroism, infra-red, fluorescence, mass spectrometry, and molecular docking. CPC was hydrolyzed using 7.09 U/mg protein of each enzyme at their optimal hydrolytic conditions for 3 h as follows: papain (pH 6.6, 60 °C), dispase (pH 6.6, 50 °C), and trypsin (pH 7.8, 37 °C). The degree of hydrolysis was in the order of papain (28.4%) > dispase (20.8%) > trypsin (7.3%). The sequence of color degradation rate and total color difference (ΔE) are dispase (82.9% and 40.37), papain (72.4% and 24.70), and trypsin (58.7% and 25.43). The hydrolyzed peptides were of diverse sequence length ranging from 8 to 9 residues (papain), 7-12 residues (dispase), and 9-63 residues (trypsin). Molecular docking studies showed that key amino acid residues in the peptides interacting with chromophore. Amino acid residues such as Arg86, Asp87, Tyr97, Asp152, Phe164, Ala167, and Val171 are crucial in hydrogen bonding interaction. These results indicate that the color properties of CPC might associate with chromopeptide sequences and their non-covalent interactions.

Molecular interaction of cyanidin-3-O-glucoside with ovalbumin: insights from spectroscopic, molecular docking and in vitro digestive studies.

Anthocyanins bound with proteins have influence on the digestibility of proteins. In this study, the interaction between cyanidin-3-O-glucoside (C3G) and ovalbumin (OVA) was investigated through multi-spectroscopy and molecular docking. The interactive effect of C3G on OVA digestibility was estimated by an in vitro digestion model. Fluorescence studies indicated that C3G quenched the fluorescence of OVA in a static mode, where the binding constants (Ka) at 298, 308 and 318 K were above 105 M-1, indicating a strong binding affinity of C3G with OVA. The negative values of thermodynamic parameters (ΔG, ΔH and ΔS) revealed a spontaneous binding process, where hydrogen and van der Waals forces were involved in stabilizing the OVA-C3G complex. The secondary structure of OVA was modified by C3G binding, with augmented ß-sheet (21.5%-25.1%) and diminished α-helix (23.3%-21.6%). Besides, C3G substantially inhibited the digestion of OVA in pancreatin solution whereas it had negligible effect on pepsin. Furthermore, molecular docking and cleavage site prediction studies showed that the hydrogen binding sites of C3G are not near to the cleavage sites of pepsin but partially overlap trypsin cleavages sites in OVA, which might lead to an inhibited effect on pancreatic digestion. These results provide a better understanding of the anthocyanin-protein interactions and their effect on the protein digestibility.Communicated by Ramaswamy H. Sarma.

Spectroscopic and molecular modelling studies on glycation modified bovine serum albumin with cyanidin-3-O-glucoside.

In this study, we report the glycation mediated effect of bovine serum albumin (BSA) on the molecular interaction mechanism of cyanidin-3-O-glucoside (C3G) by molecular modelling, Uv-visible spectroscopy, transmission electron microscopy (TEM), fluorescence spectroscopy, and circular dichroism spectroscopy studies. The structures of advanced glycation end-products (AGEs) modified BSA were modelled, energy minimized and analyzed for binding affinity by molecular docking studies using Autodock Vina. Glycation experiments are carried out using glucose and methylglyoxal to validate the molecular modelling results on the interaction of modified BSA with C3G. The modified structures were characterized by reduction in the binding pocket volume, surface, depth, hydrophobicity, and hydrogen bond donors/acceptors. Arg-194, Arg-196, Arg-198, Arg-217, Arg-409, Lys-114, Lys-116, Lys-204, Lys 221, and Lys-439 were found to be crucial in the context of glycation of BSA. TEM images represented the formation of unique globular aggregates in the event of glycation. Uv-visible spectroscopic studies showed the formation of new chromophores between 300 and 400 nm in the event of glycation. Fluorescence quenching was observed in a differential manner in the presence of C3G on glycation modified BSA. Circular dichroism studies suggested the loss of helical structure and formation of ß-sheeted structure upon glycation, but subsequent C3G binding has resulted in the increase towards helical structure. Our findings suggested that drug binding affinity has been certainly impaired due to glycation and subsequent AGE modification. Arg-p modification has more austere impact on the structure and would affect the binding properties. We conclude that C3G had differential modulation of binding properties on glycated BSA which can help to protect the stability and bioavailability that has been impaired due to glycation mediated structural changes.

Cyanidin-3-O-glucoside functions like chemical chaperone and attenuates the glycation mediated amyloid formation in albumin.

In this study, chemical chaperone like function of cyanidin-3-O-glucoside (C3G) was investigated through fluorescence spectroscopy, UV-visible spectroscopy, circular dichroism spectroscopy, confocal microscopy, scanning electron microscopy and molecular docking studies. Early and advanced glycation inhibitory effect was evaluated by fluorescence spectroscopy and agarose gel electrophoresis. Amyloids were investigated based on their propensity to bind Congo Red (CR) and Thioflavin T (ThT) by multiple microscopic approaches. Circular dichroism studies were used to analyze the changes in the secondary structure due to glycation. C3G effectively inhibited early and advanced glycation by masking like function, carbonyl scavenging and chemical chaperone activity. C3G had molecular interaction with Glu186, Arg427, Ser428, Lys431, Arg435, and Arg458 of BSA. Based on the microscopic analysis, it is evident that C3G can inhibit protein aggregation and amyloid formation. Circular dichroism studies suggested that glycation had resulted in augmented ß-sheet propensity, whereas C3G had a protective effect on the helical conformation of BSA. We conclude that C3G has a chemical chaperone like function on the event of glycation mediated amyloid formation in BSA.

Spectrofluorimetric and molecular docking studies on the interaction of cyanidin-3-O-glucoside with whey protein, ß-lactoglobulin.

The interaction of ß-Lactoglobulin (ß-Lg) with cyanidin-3-O-glucoside (C3G) was characterized using fluorescence, circular dichroism spectroscopy, and docking studies under physiological conditions. Fluorescence studies showed that ß-Lg has a strong binding affinity for C3G via hydrophobic interaction with the binding constant, Ka, of 3.14×104M-1 at 298K. The secondary structure of ß-Lg displayed an increase in the major structure of ß-sheet upon binding with C3G, whereas a decrease in the minor structure of α-helix was also observed. In addition, evidenced by near UV-CD, the interaction also disrupted the environments of Trp residues. The molecular docking results illustrated that both hydrogen bonding and the hydrophobic interaction are involved as an acting force during the binding process. These results may contribute to a better understanding over the enhanced physicochemical proprieties of anthocyanins due to the complexation with milk proteins.

Linolenic acid prevents early and advanced glycation end-products (AGEs) modification of albumin.

In this study, we report the protective effects of linolenic acid towards the formation of early (HbA1c) and advanced glycation end-products (AGEs) based on fluorescence, circular dichroism, confocal microscopy and molecular interaction studies. Linolenic acid was found to be a potent inhibitor of AGEs formed by both glucose and fructose. The HbA1c (early glycation product) level was found to be reduced to 7.4% when compared to glycated control (8.4%). Similarly, linolenic acid also inhibited the methylglyoxal mediated AGEs formation. Circular dichroism spectroscopy studies suggested that the protective effect of linolenic acid for the helical structure of albumin. The molecular interaction studies showed that linolenic acid interacts with arginine residues of albumin with high affinity. Results suggested linolenic acid to be a potent antiglycation compound and also it could be a better lead compound for AGE inhibition.

Aspartic acid functions as carbonyl trapper to inhibit the formation of advanced glycation end products by chemical chaperone activity.

Advanced glycation end products (AGEs) were implicated in pathology of numerous diseases. In this study, we present the bioactivity of aspartic acid (Asp) to inhibit the AGEs. Hemoglobin and bovine serum albumin (BSA) were glycated with glucose, fructose, and ribose in the presence and absence of Asp (100-200 µM). HbA1c inhibition was investigated using human blood and characterized by micro-column ion exchange chromatography. The effect of methyl glyoxal (MG) on hemoglobin and BSA was evaluated by fluorescence spectroscopy and gel electrophoresis. The effect of MG on red blood cells morphology was characterized by scanning electron micrographs. Molecular docking was performed on BSA with Asp. Asp is capable of inhibiting the formation of fluorescent AGEs by reacting with the reducing sugars. The presence of Asp as supplement in whole blood reduced the HbA1c% from 8.8 to 6.1. The presence of MG showed an increase in fluorescence and the presence of Asp inhibited the glycation thereby the fluorescence was quenched. MG also affected the electrophoretic mobility of hemoglobin and BSA by forming high molecular weight aggregates. Normal RBCs showed typical biconcave shape. MG modified RBCs showed twisted and elongated shape whereas the presence of ASP tends to protect RBC from twisting. Asp interacted with arginine residues of bovine serum albumin particularly ARG 194, ARG 198, and ARG 217 thereby stabilized the protein complex. We conclude that Asp has dual functions as a chemical chaperone to stabilize protein and as a dicarbonyl trapper, and thereby it can prevent the complications caused by glycation.