Nutraceutical phycocyanobilin binding to catalase protects the pigment from oxidation without affecting catalytic activity.

Phycocyanobilin is a dark blue linear tetrapyrrole chromophore covalently attached to protein subunits of phycobiliproteins present in the light-harvesting complexes of the cyanobacteria Arthrospira platensis (Spirulina "superfood"). It shows exceptional health-promoting properties and emerging use in various fields of bioscience and industry. This study aims to examine the mutual impact of phycocyanobilin interactions with catalase, a life-essential antioxidant enzyme. Fluorescence quenching experiments demonstrated moderate binding (Ka of 3.9 × 104 M-1 at 25 °C; n = 0.89) (static type), while van't Hoff plot points to an enthalpically driven ligand binding (ΔG = -28.2 kJ mol-1; ΔH = -41.9 kJ mol-1). No significant changes in protein secondary structures (α-helix content ~22%) and thermal protein stability in terms of enzyme tetramer subunits (Tm ~ 64 °C) were detected upon ligand binding. Alterations in the tertiary catalase structure were found without adverse effects on enzyme activity (~2 × 106 IU/mL). The docking study results indicated that the ligand most likely binds to amino acid residues (Asn141, Arg 362, Tyr369 and Asn384) near the cavity between the enzyme homotetramer subunits not related to the active site. Finally, complex formation protects the pigment from free-radical induced oxidation (bleaching), suggesting possible prolongation of its half-life and bioactivity in vivo if bound to catalase.

Stabilization of apo α-lactalbumin by binding of epigallocatechin-3-gallate: Experimental and molecular dynamics study.

α-Lactalbumin (ALA) is a Ca2+-binding protein which constitutes up to 20% of whey protein. At acidic pH, and in the apo-state at elevated temperatures, ALA is the classic 'molten globule' (MG). This study examined epigallocatechin-3-gallate (EGCG) binding to ALA in its apo form (apoALA) and stabilizing effect on protein structure thereof. EGCG binds to apoALA in both native and MG state. The complex of EGCG and ALA is more stable to thermal denaturation. The docking analysis and molecular dynamic simulation (MDS) showed that Ca2+ removal decreased conformational stability of ALA, because of the local destabilization of Ca2+-binding region. EGCG binding to apoALA increases its stability by reverting of conformation and stability of Ca2+-binding region. Therefore, EGCG-induced thermal stability of apoALA is based on increased apoALA conformational rigidity. This study implies that during gastric digestion of tea with milk EGCG would remain bound to ALA, albeit in the Ca2+-free form.

Covalent binding of food-derived blue pigment phycocyanobilin to bovine ß-lactoglobulin under physiological conditions.

In this study, we investigated structural aspects of covalent binding of food derived blue pigment phycocyanobilin (PCB) to bovine ß-lactoglobulin (BLG), major whey protein, by spectroscopic, electrophoretic, mass spectrometry and computational methods. At physiological pH (7.2), we found that covalent pigment binding via free cysteine residue is slow (ka = 0.065 min-1), of moderate affinity (Ka = 4 × 104 M-1), and stereo-selective. Binding also occurs at a broad pH range and under simulated gastrointestinal conditions. Adduct formation rises with pH, and in concentrated urea (ka = 0.101 min-1). The BLG-PCB adduct has slightly altered secondary and tertiary protein structure, and bound PCB has higher fluorescence and more stretched conformation than free chromophore. Combination of steered molecular dynamic for disulfide exchange, non-covalent and covalent docking, favours Cys119 residue in protein calyx as target for covalent BLG-PCB adduct formation. Our results suggest that this adduct can serve as delivery system of bioactive PCB.

Characterization and effects of binding of food-derived bioactive phycocyanobilin to bovine serum albumin.

Phycocyanobilin (PCB) is a blue tetrapyrrole chromophore of C-phycocyanin, the main protein of the microalga Spirulina, with numerous proven health-related benefits. We examined binding of PCB to bovine serum albumin (BSA) and how it affects protein and ligand stability. Protein fluorescence quenching and microscale thermophoresis demonstrated high-affinity binding (Ka=2×106M-1). Spectroscopic titration with molecular docking analysis revealed two binding sites on BSA, at the inter-domain cleft and at subdomain IB, while CD spectroscopy indicated stereo-selective binding of the P conformer of the pigment to the protein. The PCB protein complex showed increased thermal stability. Although complex formation partly masked the antioxidant properties of PCB and BSA, a mutually protective effect against free radical-induced oxidation was found. BSA could be suitable for delivery of PCB as a food colorant or bioactive component. Our results also highlight subtle differences between PCB binding to bovine vs. human serum albumin.

Stabilization of Human Serum Albumin by the Binding of Phycocyanobilin, a Bioactive Chromophore of Blue-Green Alga Spirulina: Molecular Dynamics and Experimental Study.

Phycocyanobilin (PCB) binds with high affinity (2.2 x 106 M-1 at 25°C) to human serum albumin (HSA) at sites located in IB and IIA subdomains. The aim of this study was to examine effects of PCB binding on protein conformation and stability. Using 300 ns molecular dynamics (MD) simulations, UV-VIS spectrophotometry, CD, FT-IR, spectrofluorimetry, thermal denaturation and susceptibility to trypsin digestion, we studied the effects of PCB binding on the stability and rigidity of HSA, as well as the conformational changes in PCB itself upon binding to the protein. MD simulation results demonstrated that HSA with PCB bound at any of the two sites showed greater rigidity and lower overall and individual domain flexibility compared to free HSA. Experimental data demonstrated an increase in the α-helical content of the protein and thermal and proteolytic stability upon ligand binding. PCB bound to HSA undergoes a conformational change to a more elongated conformation in the binding pockets of HSA. PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis. The results from this work explain at molecular level, conformational changes and stabilization of HSA structure upon ligand binding. The resultant increased thermal and proteolytic stability of HSA may provide greater longevity to HSA in plasma.

Conformational stability of digestion-resistant peptides of peanut conglutins reveals the molecular basis of their allergenicity.

Conglutins represent the major peanut allergens and are renowned for their resistance to gastro-intestinal digestion. Our aim was to characterize the digestion-resistant peptides (DRPs) of conglutins by biochemical and biophysical methods followed by a molecular dynamics simulation in order to better understand the molecular basis of food protein allergenicity. We have mapped proteolysis sites at the N- and C-termini and at a limited internal segment, while other potential proteolysis sites remained unaffected. Molecular dynamics simulation showed that proteolysis only occurred in the vibrant regions of the proteins. DRPs appeared to be conformationally stable as intact conglutins. Also, the overall secondary structure and IgE-binding potency of DRPs was comparable to that of intact conglutins. The stability of conglutins toward gastro-intestinal digestion, combined with the conformational stability of the resulting DRPs provide conditions for optimal exposure to the intestinal immune system, providing an explanation for the extraordinary allergenicity of peanut conglutins.