Eric Fouquet

Erick J. Dufourc

Isabelle Pianet

Olivier Cala

Sandy Fabre

Comptes Rendus Chimie

Full paper / MémoireNMR of human saliva protein/wine tannin complexes. Towardsdeciphering astringency with physico-chemical toolsC. R. Chimie 13 (2010) 449–452Contents lists available at ScienceDirectComptes Rendus Chimiewww.sciencedi rec t .comOlivier Cala a,b, Sandy Fabre a, Eric Fouquet a, Erick J. Dufourc b, Isabelle Pianet a,*a ISM-UMR 5255, CNRS, université Bordeaux 1, 351, cours de la Libération 33400 Talence, Franceb CBMN-UMR 5248, CNRS, université Bordeaux1, ENITAB, 2, rue Robert-Escarpit, 33600 Pessac, FranceA R T I C L E I N F OArticle history:Received 4 May 2009Accepted after revision 12 January 2010Available online 23 February 2010Keywords:NMRDOSYInteractionAstringencyMots clés :RMNDOSYInteractionAstringenceA B S T R A C TIn the present work, we used NMR (chemical shift variations and diffusion measurement)to determine the affinity and the number of sites of four different 4-8 procyanidin dimersfor a model PRP peptide. A number of three binding sites has been identified for everytannin, but significant affinity differences are highlighted from one to another dimer(a factor 10 approximately) that could be related to their specific 3D-structure. 2010 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.R É S U M ÉDans ce travail, nous avons utilisé la RMN (en particulier, nous avons suivi les variations dedéplacements chimiques et de coefficient de diffusion) pour déterminer l’affinité et lenombre de sites de fixation de quatre différents dimères de procyanidines pour un peptidemodèle de PRP. Nous avons pu ainsi identifier trois sites différents de fixation, quel que soitle dimère. En revanche, des différences d’affinité significatives ont pu être mises enévidence d’un dimère à un autre (un facteur dix environ) et ces différences peuvent êtreexpliquées par leur structure 3D spécifique. 2010 Académie des sciences. Publié par Elsevier Masson SAS. Tous droits réservés.1. IntroductionAstringency is considered as a proof of red wine quality.It appears as a dry, rough or even pucker sensation in theentire mouth while tasting [1]. A consensus of opinionsuggests that this sensation is the result of a recognitionprocess occurring between tannins and saliva proteins [2–8]. Wine tannins are mainly issued from the solid part ofthe grape, skins, seeds and stems. They are transferred towine during wine-making and can reach a concentrationup to 4 g/l in Bordeaux red wines. They are oligomers offlavan-3-ol also called Procyanindins [9] and represent alarge family owing to their two distinct constitutive units,the two types of linkage possible in between two units (4-6* Corresponding author.E-mail address: i.pianet@ism.u-bordeaux1.fr (I. Pianet).1631-0748/$ – see front matter  2010 Académie des sciences. Published by Edoi:10.1016/j.crci.2010.01.003or 4-8) and the number of constitutive units or degree ofpolymerization (up to 13 constitutive units were found inred wine [10]).Human parotid saliva proteins are composed up to 70%of Proline-Rich Proteins (PRPs) divided in three groupsdepending on their specific functions, glycolysed, acid andbasic PRPs [11]. Only basic PRPs are known to bindpolyphenols [12,13] and this specific binding results in thetactile astringent sensation. As their name let us suggest,PRPs are mostly composed of Proline (40%) but also ofglycine and glutamine, these three amino acids corre-sponding up to 80% of the total amino acids content [14].They are organized in two types of repeated sequences offive (PQGPP) and nine amino acids (PPGKPQGPP), whichconfer on them an extended structure open, to interactwith tannins [15]. In the present work, we monitor theinteractions of the four different 4-8 linked procyanidindimers (Scheme 1) with a peptide of 14 residus (IB7-14)lsevier Masson SAS. All rights reserved.Scheme 1. Structure of the 4 4-8 procyanidin dimers studied.O. Cala et al. / C. R. Chimie 13 (2010) 449–452450representing a PRP widespread consensus sequence usingdifferent types of NMR experiments. Following the peptideproton chemical shift or even the diffusion coefficientvariations induced by adding the different procyanidinsgives rise to some physico-chemical parameters of interest[16]: the stoichiometry of the complex formed, theinteraction sites and the affinity constant of the complexformation. The differences observed in between tanninscould be used to establish an affinity scale of tannins, basedon the hypothesis that the astringency sensation is directlylinked to the affinity of a tannin towards a saliva PRP.2. Materials and methods2.1. SynthesisThe chemical synthesis of the peptide IB7-14(SPPGKPQGPPPQGG) was performed on an Applied Bio-systems petide synthetizer 433A by the FastMoc methodsusing a method previously described [17]. Procyanidinsdimers B1, B2, B3, and B4 (Scheme 1) were synthesizedusing the previously described methods [18].2.2. Sample preparationA 1 mM protein sample was dissolved in a water/D2O/EtOD: 80/8/12 mixture, with 5 mM deutered acetic acid,pH 3.5 to mimic wine conditions. Tannins were progres-sively added to the protein sample to scan a tanninconcentration ranging from 1 to 7 mM. In order to keepconstant the protein concentration, tannins were justadded under a powder form after being dissolved in water,aliquoted and lyophilized. Using such a procedure, thesame 5 mm NMR tube was used with the 1 mM proteinsolution and lyophilized tannin was added for each ratio.2.3. NMR experimentsSpectra were recorded at 298 K on a Bruker DXP 400spectrometer equipped with a 5 mm gradient inversebroadband probe with 2H lock. All proton chemical shiftsare given with respect to TSP as an internal reference sothat it also can be used for quantification purpose. 1Dspectra were recorded using a Watergate Sequence inorder to suppress efficiently the water resonance [19].Diffusion measurements were performed using the Stejkaland Tanner pulse sequence [20] using the followingparameters: spectral width = 12 ppm; NS = 64; recyclingdelay = 3 s; intergradient delay D = 150 ms; gradient pulseduration d = 2 ms. The pulse gradients (G) were incremen-ted from 2 to 95% of the maximum gradient strength in alinear ramp in 16 steps. The diffusion coefficient, D, can beobtained by fitting a specific resonance area, I, obtained atdifferent gradient powers (G) using the following equa-tion:I = eg2G2Dd2 (Dd/3).NMR data analysis: For titration experiments, chemicalshifts variations of some protons of the peptide wereanalyzed as a function of procyanidin dimer concentrationusing the equation previously described for a multisitemodel [21]:Aobs ¼12Amax ð1þ Kd=n ½Pi þ ½Ti=n½PiÞ fð1þ Kd=n ½Pi þ ½Ti=n ½PiÞ2  4 ½Ti=n ½Pig1=2(2)where Aobs is Ddi, the chemical shift variation (ppm) orDDobs, the diffusion coefficient variation, Amax is Ddmax (orDDmax), the chemical shift (diffusion D) differencebetween the chemical shift (or D) of the protein aloneand the protein saturated in tannins, Kd is the dissociationconstant expressed in M, [Ti], the total concentration ofpolyphenol able to fix the peptide (M) by taking intoaccount their autoassociation (the Ka of the autoassocia-tion process has been evaluated at around 6 M1, [22]),[P0], the total concentration of peptide (M), and n, thenumber of polyphenol binding sites. Kd, n and Ddmax werecalculated using a least-squares-fitting routine within thesoftware program EXCEL.3. Results3.1. Chemical shift variationsFig. 1 shows the chemical shift variations of the peptideNH resonances when procyanidin B1 is progressivelyadded to the peptide solution. All the peptide NMR protonsignals have been already assigned [16]. Significantchemical shifts are observed for all the NH resonancesbut the effect is exemplified on the resonances of Glycine13 and 14 (G13, G14) NH. Proline 2, 9 and 10 (P2, P9, P10)Ha resonances are also affected by the presence ofFig. 1. 1H spectra of a 1 mM peptide IB7-14 solution recorded withdifferent procyanidin contents (from 0 to 7 mM).Fig. 2. Chemical shift variation of G13 NH of the peptide (1 mM) whendifferent procyanidins are added.Fig. 3. Diffusion coefficient evolution of 1 mM IB714 peptide (D, 10-10m2 s-1) with increasing tannins concentrations. Diamond (B1) triangle(B2) square (B3) and circle (B4) are the experimental data; Solid linesrepresent the best fit using eq. (1) with Kd = 3.5 mM, n = 2.6, (Dmax = 0.75and (2 = 2.2 10-2 for B1; Kd = 0.8 mM, n = 3.6, (Dmax = 1.1 and (2 = 0.8 10-2for B2; Kd = 8.5 mM, n = 3.3, (Dmax = 0.9 and (2 = 0.9 10-2 for B3;Kd = 1.4 mM, n = 3.7, (Dmax = 1.1 and (2 = 4.2 10-2 for B4.O. Cala et al. / C. R. Chimie 13 (2010) 449–452 451procyanidin dimers. This suggests that the fixation sitesare probably the same for each tannin. These chemical shiftvariations were fitted using Eq. (2) describing the fixationprocess of n tannins on a protein with the same affinity(i.e., the same Kd for every tannin), as previously described[23]. Fig. 2 displays G13 NH chemical shift variationsobserved when B1 (diamond), B2 (triangle), B3 (circle) orB4 (square) are progressively added (ranging from 0 to7 mM) to the 1 mM peptide solution, and their fitting(lines), Kd, n, and Ddmax being the adjustable parameters.Kd and n values obtained for different protons (G13 andG14 NH, P2, P9 and P10 Ha) were averaged and arereported on Table 1.Table 1Dissociation constant (Kd), and number of tannin binding sites (n) wereobtained from the fit of experimental chemical shift variations of G13 andG14 NH, P2, P9 and P10 Ha, and from the diffusion coefficients variationof the petide using eq. (2). The different Kd and n values obtained for onetannin were averaged are reported SD.Procyanidins Kd (mM) nB1 2.9 1.4 2.6 0.4B2 1.2 0.4 3.6 0.5B3 8.0 0.9 3.3 0.5B4 1.4 0.4 3.7 0.3As shown by the standard deviation measures, valueswere obtained with a better accuracy than valuespreviously reported [16]. This is probably due to a tidycontrol of peptide vs tannin concentration all along thetitration. This accuracy is of crucial importance because itallows us to have sufficient confidence in Kd and n valuesand in their differences to rank tannins with respect totheir affinity towards the PRP peptide.3.2. Diffusion measurementsNMR diffusion experiments are powerful for detectingthe association of small molecules with a host moleculenot exceeding a few kDa in molecular weight. Indeed, insuch a case it is easy to follow changes in the diffusionspeed of the host all along the binding of guest molecules[24] for determining both Kd and the number of fixationsites. Thus, following changes of the 1.3 kDa PRP peptidediffusion rate with increased tannin content (dimermolecular weight: 479 Da) is possible as shown inFig. 3. A progressive decrease of D is observed as thetannin content increases, in agreement with the increase ofthe overall complex size. Experimental data for each tanninwere fitted using Eq. (2) (in this case, Aobs = Dobs) andallowed to measure Kd and n measurements. The valuesfound for each tannin were used to calculate the averagedvalues reported on Table 1. D values evolve fromapproximately 3.0 1010 m2.s1 for the peptide alone to2.0 1010 m2.s1 when complexed with procyanidindimers. This corresponds to a change in the hydrodynamicradius rH from 6.8 for the peptide alone to 8.2 Å for thepetide complexed with at least three procyanidin dimers.4. DiscussionIn the present work, the interactions between fourdifferent procyanidin 4-8 dimers and the human salivaprotein fragment IB714 were monitored by1H NMRspectroscopy. Using both chemical shift and diffusioncoefficient, titration information on the stoichiometry ofO. Cala et al. / C. R. Chimie 13 (2010) 449–452452the complex and dissociation constants were determinedusing a multisite model of Protein-ligand for which eachligand exhibits the same affinity toward the protein aspreviously described [16,21]. Monitoring both chemicalshift and diffusion variations gives rise to some crucialinformation. First, some information is obtained about thefixation sites: the number of fixation sites has beenevaluated to three whatever the procyanidin dimerconsidered, and if the peptide protons that undergo thegreater chemical shift variations can be assimilated to thefixation sites, three different sites can be identified: P2, P9-P10 and G13-G14. These sites are the same for each tannin.Second, some information is collected about tannin-salivaprotein affinity. Dissociation constants (Kd) are obtained byfitting experimental data and give rise to value measure-ment with a good accuracy. These values vary from 1.2 to8 mM, despite the tenuous chemical structure differencesin between the four dimers studied. B2 and B4 dimerspresent a higher affinity than B1 and, a fortiori, B3 towardsthe saliva peptide IB7-14, leading to rank tannins as regardto their affinity. For these measurement, the procyanidinself-association process has been taken into account, theassociation constant of this phenomenon being closed to7 M1, whatever the procyanidin dimer considered [22].Nevertheless, it is noteworthy that this phenomenon isfavored at higher concentration than those we used: thecritical micellar/aggregation concentration for dimers isabove 17 mM, and titration experiments were done atlower procyanidin dimers concentrations (under 7 mM).Of interest is to compare these affinity differences to3D-structure of procyanidin dimers. 4-8 procyanidindimers are known to exist upon two rotameric forms: acompact form, which is largely dominant for B1 and B3(95%) and an extended form which exist, at a level up to24% for B4 and, up to 45% for B2 [25]. This let us suggestthat the extended conformation must facilitate theapproach or/and the fixation of the tannin to the peptide:the more the extended conformation is preferred, thehigher the affinity towards the PRP peptide is.AcknowledgementsThis work has been supported by the Conseil inter-professionnel de Bordeaux (CIVB), the CNRS, the université deBordeaux 1. No conflict of interest exists for all the co-authors.References[1] E.C. Bate-Smith, Foods 23 (1954) 124.[2] M. A. Joslyn, J. L. Goldstein, Astringency of fruit and fruit products inrelation to phenolic content, New York Academic Press, M. E. Mrack,G.F. Stewart (Eds), Chichester, 1964.[3] E.C. Bate-Smith, Phytochemistry 12 (1973) 907.[4] V.L. Singleton, A.C. Noble, Wine flavor and phenolic substances, Amer-ican Chemical Society, Washington, 1976.[5] A.E. Hagerman, L.G. Butler, J Biol Chem 256 (1981) 4494.[6] P. Sarni-Manchado, V. Cheynier, M. Moutounet, 2nd Internationalelectronic conference on synthetic organic chemistry, 1998.[7] V. De Freitas, N. Mateus, J Agric Food Chem 49 (2001) 940.[8] T. Hofmann, A. Glabasnia, B. Schwarz, K.N. Wisman, K.A. Gangwer, A.E.Hagerman, J Agric Food Chem 54 (2006) 9503.[9] E. Haslam, Practical Polyphenolics, Cambridge University Press, Cam-bridge UK, 1998.[10] M.F. Nonier, C. Absalon, N. Vivas, N.V. de Gaulejac, J Chromatogr A 1033(2004) 291.[11] A. Bennick, Mol Cell Biochem 45 (1982) 83.[12] H. Mehansho, L.G. Butler, D.M. Carlson, Annu Rev Nutr 7 (1987) 423.[13] Y. Lu, A. Bennick, Arch Oral Biol 43 (1998) 717.[14] D.L. Kauffman, P.J. Keller, Arch Oral Biol 24 (1979) 249.[15] N.J. Murray, M.P. Williamson, Eur J Biochem (1994) 219.[16] C. Simon, K. Barathieu, M. Laguerre, J.M. Schmitter, E. Fouquet, I. Pianet,E.J. Dufourc, Biochemistry 42 (2003) 10385.[17] C. Simon, I. Pianet, E.J. Dufourc, J Pept Sci 9 (2003) 125.[18] I. Tarascou, K. Barathieu, Y. André, I. Pianet, E.J. Dufourc, E. 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NMR of human saliva protein/wine tannin complexes. Towards deciphering astringency with physico-chemical tools

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NMR of human saliva protein/wine tannin complexes. Towards deciphering astringency with physico-chemical tools

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