Study of the interaction between salivary proline‐rich proteins and a polyphenol by ¹H‐NMR spectroscopy

Abstract

The interaction between salivary proline-rich proteins and plant polyphenols (tannins) in the oral cavity and their subsequent precipitation influences the taste, texture and nutritional value of food ; it is thought to be responsible for the astringency of many foods and beverages. To investigate the interaction, two-dimensional 'H-NMR studies have been carried out on the binding of a representative polyphenol, pentagalloyl glucose, to two synthetic peptides (19 and 22 residues in length) that are typical of the repeat sequence of mouse salivary proline-rich protein MP5. Intermolecular nuclear Overhauser effects and chemical shift changes show that the main binding sites on the peptides are proline residues together with the preceding amide bond and amino acid. The interaction is principally a hydrophobic association between a galloyl ring and the pyrrolidine ring face containing the Ca proton, but secondary hydrogen-bonding effects help to stabilise the complex. Very similar interactions are seen for both peptides. The conformation of the peptides remains extended on binding. The chemical shift changes seen for many of the peptide protons can be fitted to a simple bindmg curve with dissociation constant of around 40 mM, but some protons show evidence of cooperative binding involving several galloyl groups. Higher concentrations of pentagalloyl glucose lead to a reduced off-rate from the complex and eventual precipitation which implies that precipitation is caused by a kinetic competition between aggregation and dissociation of the complex.Polyphenols (vegetable tannins) are a major constituent of plants, including many that are consumed widely by humans. They are present in foods (e.g. sorghum, millet, fruit, berries, cocoa), beverages (e.g. coffee, tea, beer, wine, cider), folk medicines and herbal remehes. Polyphenols are charactensed by a multiplicity of phenolic groups and have molecular masses in the range 500-5000Da. They have been classified into two groups: the hydrolysable tannins, which are esters, usually with D-glucose, of gallic acid and its derivatives, and the condensed tannins (proanthocyanidins) which are oligomers of the flavan-3-01 skeleton, e.g. epicatechin [l]. Their biological effects are wide-ranging and include inhibition of gut enzymes, inhibition of non-haem iron absorption, influencing the taste and reducing the nutritional value of food, oesophageal cancer, and the effect of astringency, i.e. a feeling of extreme dryness, puckeriness and constriction Abbreviations. PRP, proline-rich protein; Gall,Glc, 1,2,3,4,6-penta-U-galloyl-/I-D-glucopyranose; (C2H3)2S0, (Z&)dimethyl sulphoxide ; ROESY, two-dimensional rotating-frame nuclear Overhauser effect spectroscopy ; TOCSY, two-dimensional total correlation spectroscopy ; NOESY, two-dimensional nuclear Overhauser effect spectroscopy; AS-, maximum chemical shift change; ROE, rotating frame nuclear Overhauser effect; 35HN,. three-bond coupling constant between NH and CaH; AS/AT, chemical shift temperature coefficient; dm, sequential NOE conn...

Association of Tannins and Related Polyphenols with the Cyclic Peptide Gramicidin S.

“…It was also suggested that the unique characteristics of the Pro residue, its flat and rigid structure and hydrophobicity, is important in the tannin-protein interactions at the bturn structure and not only in linear peptides. 1,2) …”

“…Recently, the preferential association of tannins with proline (Pro) residues in linear peptides was reported. 1,2) Pro residues disrupt both a-helix and b sheet conformation in the peptide secondary structure and commonly occurs on the surfaces of proteins. 3) Since Pro residues are frequently found near protein-protein interaction sites, 4) the preferential interaction of tannins with Pro residues may be related to the broad biological activities of tannins including enzyme inhibition.…”

Tannin interactions with a full‐length human salivary proline‐rich protein display a stronger affinity than with single proline‐rich repeats

“…The upfield changes in chemical shift seen on titration of the protein with tannins can be ascribed to ring current shifts, caused largely by face-to-face stacking of the prolyl rings of the protein with the galloyl rings of the tannins [5]. Each proton within the protein experiences different upfield shifts due to local tannin binding, and therefore the curve fitting procedure essentially yields individual microscopic dissociation constants for each site in the protein.…”

“…where ASi is the change in chemical shift, [P]i is the total concentration of protein, [T]i is the total concentration of polyphenol, [P] This expression assumes a 1:1 binding interaction at each fitted proton, which has been justified elsewhere for PRP/polyphenol ass()-ciations [5]. The curve fittings were performed using EXCEL (Microsoft Corp.).…”

“…Tannins have a range of harmful effects including the inhibition of digestive enzymes [4], and their complexation with salivary PRPs causes them to be precipitated and excreted in a harmless form. In a previous study [5], we have investigated the binding of a tannin to a single PRP repeat sequence and have shown that the binding is mainly a hydrophobic interaction between proline rings and the galloyl rings of the tannin. Here, we have repeated the binding investigation using [3-1,3,6-tri-O-galloyl-Dglucopyranose (TriGG), [~-l,2,4,6-tetra-O-galloyl-D-glucopyranose (TetraGG), 13-1,2,3,4,6-penta-O-galloyl-D-glucopyranose (PentaGG) and (-)-epicatechin complexing with a fulllength human salivary PRP, and the results indicate that the association observed is significantly stronger than with the single repeat sequence.…”