Monitoring the Interactions of a Ternary Complex Using NMR

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Monitoring The Interactions of a Ternary Complex using NMR spectroscopy: The case of Sugars, Polyphenols and Proteins Benoit Faurie, Erick Joel Dufourc, Michel Laguerre, and Isabelle Pianet Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03911 • Publication Date (Web): 10 Nov 2016 Downloaded from http://pubs.acs.org on November 17, 2016

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Analytical Chemistry

Monitoring The Interactions of a Ternary Complex using NMR spectroscopy: The case of Sugars, Polyphenols and Proteins. Benoit Faurie1,2, Erick J. Dufourc2, Michel Laguerre2 and Isabelle Pianet1*

1-CESAMO-Institut des Sciences Moléculaires- UMR 5255, Université de Bordeaux, CNRS, 351 cours de la Libération 33405 Talence France ; 2-Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN- UMR 5248), Université de Bordeaux, CNRS, INP Bordeaux, Allée Geoffroy Saint-Hilaire 33600 Pessac, France. * to whom correspondence should be sent : [email protected]

KEYWORDS. Polyphenols, Polysaccharides, Proline-Rich Protein, Interactions, NMR, Molecular Modelling, Dynamic Light Scattering.

ABSTRACT. Gaining insight into intermolecular interactions between multiple species is possible at an atomic level by looking at different parameters using different NMR techniques. In the specific case of the astringency sensation, in which at least three molecular species are involved, different NMR techniques combined with dynamic light scattering and molecular modelling

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contribute to decipher the role of each component in the interaction mode and to assess the thermodynamic parameters governing this complex interaction. The binding process between a saliva peptide, a polyphenol and polysaccharides was monitored by following 1H chemical shift variations, changes in NMR peak areas and size of the formed complex. These NMR experiments deliver a complete picture of the association pathway, assessed by Dynamic Light Scattering and molecular dynamics simulations: all the data collected converge towards a comprehensive mode of interaction in which sugars indirectly play a role in astringency by sequestering part of the polyphenols, reducing their effective concentration to bind saliva proteins.

INTRODUCTION Monitoring the interactions of a complex in which more than two components are involved is not trivial and the methodology used to characterize such a complex, from both physical and chemical viewpoints must fulfil certain prerequisites. Notably, the sensitivity of the techniques used has to be adequate for the phenomenon to be observed, a condition easily satisfied when only two components are involved in the interaction, but which becomes more complicated with three. The specific behaviour of each component must also be known in the experimental conditions used (notably concentration, solvent, temperature). Facing such complexity, NMR appears to be a suitable technique. Numerous spectroscopic NMR parameters may serve as a gauge to understand the binding process at a molecular level, even with more than two molecular species involved.1 An example to illustrate such a case is the role played by each major constituent responsible for the sensation of astringency, saliva proteins, polyphenols and polysaccharides. Astringency is described as a tactile sensation2 governed by the interaction between polyphenols present in beverages such as wine or tea, and saliva proteins belonging to the Proline Rich family.3 It has recently been demonstrated that the interaction mode depends on the colloidal state of the


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polyphenols.4 Polyphenols bind the proline rich saliva peptides in a specific way below their Critical Micellar (or Aggregation) Concentration5, and with an affinity depending on the chemical nature of the polyphenols. Above this threshold they interact in a non-specific way leading to the precipitation of the polyphenol-protein complex. Therefore, one may wonder if there is a relationship between the colloidal behaviour of polyphenols and the perception of astringency as suggested by the varied vocabulary used by oenologists for describing this buccal sensation.

6,7

However, astringency is also greatly influenced by other components present in beverages, notably the sugar content8. In fact, the ripening of the fruit has been associated with the release of soluble fragments of pectin as the cellular structure of the fruit softens.9 For this reason the implication of polysaccharides in the sensation of astringency is commonly accepted.10 However, the way by which they act is still the subject of debate. Do they interact with polyphenols, as suggested Matsuo and Itoo, more than 30 years ago11 ? Or do they prefer to bind the protein moiety leading to a disruption of the association between polyphenols and proteins 9? Or is a more complex molecular assembly invoked, composed of the three partners, polyphenols-proteins-polysaccharides, as more recently proposed by Mateus et al.12? The chemical nature of all the partners probably intervenes in the process as proposed by Soares et al. 13 In this paper we follow several spectroscopic NMR parameters to decipher the binding network between three different molecular species: a peptide representative of saliva Proline-Rich proteins, IB7-14, a polyphenol, Epigallocatechin Gallate, and different sugars as simple as glucose or as complex as Arabic Gum or Pectin (see scheme 1 for chemical structures). The proton chemical shift changes of the different species provide access to the binding sites and the thermodynamic parameters, Diffusion-Ordered Spectroscopy (DOSY) permits us to evaluate the evolution of the size of the complex formed and High-Resolution Magic Angle Spinning NMR spectroscopy allows us to observe part of the NMR signal that escapes detection using traditional solution NMR.



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Dynamic Light Scattering and molecular modelling are also used to strengthen the model of interaction between the three molecular species.

Scheme 1. Chemical structure of the model peptide IB7-14 and polyphenol Epigallocatechin Gallate (EGCG).

MATERIAL AND METHODS

Materials. Epigallocatechin gallate (EGCG), α-D-glucose, Pectin and Arabic Gum (AG) from acacia, were purchased from Sigma-Aldrich. IB7-14 peptide (1SPPGK-PQGPPPQGG14) was synthesized in the laboratory by using the experimental conditions previously described. 14

Samples preparation. For the CMC measurement, EGCG was dispersed in a wine-like buffer composed of H2O/D2O /EtOD (80:8:12, v:v:v) and CD3COOD (5 mM) adjusted to pH 3.5. For the titration experiments, a mixture of IB7-14/Sugar or EGGC/IB7-14 with a chosen ratio was prepared in the wine-like buffer. To keep constant these concentrations, stock solutions of sugar and polyphenols were prepared and the exact quantity used for each point of the titration was subsequently lyophilised and added as a powder.


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Liquid-State NMR experiments. NMR experiments were performed at 298 K on a Bruker 700 MHz spectrometer equipped with a 5 mm TXI probe 1H/13C/15N/2H (IECB facility). All proton chemical shifts are given with respect to the residual protonated ethanol as an internal reference (two peaks at 1.0 and 3.5 ppm). 1H spectra were recorded using a single-pulse experiment with a Watergate pulse sequence15 to suppress water and using the following parameters: spectral width 12 ppm, 90° nutation angle duration 7.5 µs, 90° watergate pulse duration 9 µs, recycling delay 5s, (3s acquisition time and 2s relaxation delay to ensure the total relaxation of signals). Diffusion measurements were performed using 1H NMR bipolar pulsed gradients16 .The following parameters were used: spectral width 12 ppm, scan number 32, recycling delay 2 s, intergradient delay ∆ 200 ms; gradient pulse duration δ 3 ms; the pulsed gradients G were incremented from 2 to 95% of the maximum gradient strength (50 G.cm-1) in a linear ramp with 16 steps.

MAS NMR experiments. Magic Angle Spinning NMR was performed on a HR-MAS probe 1

H/13C with 4 mm rotor at 600 MHz (CESAMO facility). The rotor was filled with around 50 µL of

the liquid samples and the 1H spectra were recorded with the same sequence and conditions as for the liquid experiments.

NMR data analysis. The 1H chemical shift variation (∆δ) was directly determined on the 1D spectrum. For EGCG self-association, plotting (∆δ) against 1/[EGCG] leads to two straight lines with different slopes whose intercept gives the CMC value as previously described.5 The diffusion coefficient, D, was obtained by fitting the area, I, of a given resonance as a function of the amplitude of the gradient G using the equation

ln(I/I0) = -γ2G2Dδ2(∆ - δ/3)

(1)

Where γ is the proton gyromagnetic ratio and I0 the area without any gradient.



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The self-association constant of EGCG, Ka, is obtained by following the evolution of either the chemical shift (δ) or the diffusion coefficient (D) versus concentration.5 The δ/D changes were least-squares fit using the following equation:17

Aobs = Afree- [(|Amicelle- Afree|)KaT0{2/[1 + ({KaT0+1)1/2]}2]

(2)

where Aobs corresponds to the observed D/δ, Afree to the D/δ of the non-associated tannin, Amicelle to D/δ of the tannin embedded in a micelle, and T0 is the total tannin concentration. Ka, Afree and A micelle

were calculated using a least-squares-fitting routine within the software Excel, the best values

chosen were those presenting the lowest χ2. The protein-polyphenols dissociation constant Kdtp was obtained by following the chemical shift variations of the most responsive protons of the peptide (Hα P2 and P10, NH G 8, 14 and Q7) through EGCG titration in the presence or absence of different sugars (5.6 mM Glucose, 10 µM pectin- corresponding to 1.8 mM Eq. galacturonic acid, and 1 µM Arabic Gum- corresponding to 1.8 mM eq. hexose). Values were fitted using the following equation (Eq. 4) 18 Aobs=1/2∆Amax [(1+Kdtp/n[P0]+[Ti]/n[P0])–{(1+Kdtp/n[P0]+[Ti]/n[P0])2– 4[Ti]/n[P0]}1⁄2] (4) Where Aobs is the chemical shift variation (ppm) ∆δ between the chemical shift of the protein alone and the observed chemical shift; Amax is the chemical shift difference ∆δmax between the chemical shift of the protein alone and saturated with polyphenols; Kdtp is the dissociation constant (in M); [Ti] is the concentration of polyphenol (M) that can bind the peptide by taking into account their self-association (Ka determined with eq. 2); [P0] is the total concentration of peptide (M); and n is the number of polyphenols binding sites. ∆δmax, Kdtp and n were evaluated using the leastsquares-fitting routine of the Excel software (Microsoft, Redmond, WA, USA). Equation (4) was also used to evaluate the size of the micelles formed with Aobs= ∆Dobs, and Amax = Dmicelle. The micelle hydrodynamic radius was estimated from the Stokes-Einstein relationship adapted to evaluate the size of a spherical object : Dmicelle=kBT/6πηRH

(5)


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Where the viscosity, η, was measured for each solution and was quite similar from one solution to another and close to 1.3 mPa.s

The relative quantification of free sugar remaining in solution was performed on the proton spectra by using the residual signal of ethanol as an internal reference. The free sugar quantity variations (SL) were analysed as a function of [EGCG] (T0) using eq 6:

SL = -S0/2*((1+T0/S0 + Kdst/S0)-((1+ T0/S0 + Kdst/S0)2-4*T0/S0)^/2) + S0 (6) Where SL is the free sugar in solution (in M) determined by the integration of different sugars resonances with respect to an internal reference (ethanol, in the present study), S0 is the initial sugar concentration (in M), T0 is the total EGCG concentration (in M) and Kdst is the dissociation constant between sugar and EGCG (in M). Kdst was calculated using the least-squares-fitting routine of Excel (Microsoft, Redmond, WA, USA).

Dynamic Light Scattering. DLS experiments were performed on a Zetasizer (MALVERN) apparatus. Samples consisted of 2.5 µM of AG in a wine like medium in which EGCG was progressively added to reach a final concentration of 10 mM. The correlation signal decay versus time collected at a constant angle of 90° was fitted using a multiple exponential function in order to obtain the distribution of the particle sizes.

Molecular Modelling. Calculations were performed using GROMACS version 4.5 and the GROMOS96 force field (G43al). The EGCG and glucose were configured using the Dundee PRODRG2 server website version 2.5 (http://davapc1.bioch.dundee.ac.uk/prodrg/). Two molecular dynamic simulations were run: the first one with 6 glucose and 6 EGCG molecules in a water box of (100 Å)3 containing 99480 atoms sorted in 33044 residues including glucose and EGCG; the second one with 36 glucose and 36 EGCG in the same water box of (100 Å)3 containing 98451



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atoms distributed in 32121 residues including the 36 Glucose and EGCG molecules. Glucose and EGCG molecules were regularly positioned in the box. Molecular Dynamic runs were performed at constant temperature (300K) and pressure (1 bar). 10 successive runs of 10 ns were performed for a total time of 100 ns. Each system was first energy minimized with 50 cycles of the steepest descent, time step 2 fs, PME (Particle Meshed Ewald) method was on with a cubic grid (1 Å), VdW cutoff 10 Å, and frames were saved every 1000 steps. The EGCG and Glucose concentrations are equivalent to 10 and 60 mM for the two experiments.

RESULTS

1

H NMR spectroscopy:

Two series of titrations were performed, by varying the sugars concentration and leaving constant the concentration of EGCG and by varying the EGCG concentration and keeping the sugar concentration constant. In the two cases, the titrations were performed in the absence or presence of a peptide representative of Proline Rich Protein, IB7-1419, at a concentration close to 0.5 mM. For the first titration experiment, a series of 1H spectra was recorded at two different concentrations of EGCG (1 and 10 mM with respect to its CMC20) in the absence or presence of the peptide IB7-14, the concentration of Arabic gum varying from 0 to 0.5 g/L (2 µM, approx. corresponding to a 3 mM equivalent hexose concentration). The signal intensity of EGCG significantly decreases as the concentration of AG increases (Figure 1, Supplementary Figure S1a), and this phenomenon appears to be independent of the presence of the peptide as suggested by the spectrum shown in Figure 1A. However, it is noteworthy that no chemical shift variation was observed either on the polyphenol or on the peptide signals during the progressive addition of sugars.


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The same type of titration was performed with glucose. When Glucose was added to a 1 or 10 mM EGCG solution, part of the EGCG signal also disappeared but to a lesser extend (up to 10% of the EGCG signal disappeared when 50 mM of glucose was added, see supplementary Figure S1b).

Figure 1. (A) Up: 1H NMR spectra (zone corresponding to the two H4 of EGCG) of a 10 mM EGCG solution recorded in absence (black) and in presence (grey) of 0.3 g/L AG in the presence of 1 mM IB7-14 (zone showing the H ε of K5). (B) Variation of H4 area between 2.85 and 2.95ppm of EGCG with respect to the AG concentration : 1mM EGCG alone (X) or with 0.5 mM IB7-14 () ; 10 mM EGCG alone () or with 0.5 mM IB7-14 ().

For the second titration experiment, a series of 1H NMR spectra were recorded in the presence of glucose (5.6 mM), pectin (10 µM / 1.8 mM eq galacturonic acid)- or AG (1.3 µM / 1.8 mM eq. hexose) without or with the peptide IB7-14 (0.5 mM), varying the EGCG concentration from 0 to 5 mM. In these conditions, we observed a significant decrease of the sugar resonances area through the EGCG titration as displayed in Figure 2, and supplementary Figure S2: whatever the sugar added, a decrease in the signal intensity is observed, up to 10% for glucose, 28% for pectin and 50% for Arabic gum. The dissociation constant Kdtp and the number of binding sites, n, between



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EGCG and the peptide were estimated for every condition (without sugar or in the presence of pectin or AG) by following the chemical shifts of the protons of the peptide that were responsive upon addition of EGCG (Figure 3, supplementary Figure S3). As previously described for different procyanidins 4, the chemical shift variations observed through the titration exhibits two distinct phases : for the lowest EGCG concentrations (till 3mM), experimental points could be adequately fitted using eq.4, giving the physico-chemical values requested (Kdtp and n); for EGCG concentration higher than 3mM, chemical shifts evolved linearly as the polyphenol concentration increased. This phenomenon has already been described to be the consequence of non specific interactions occurring between the two species at polyphenol concentrations higher than their CMC (4.6±0.6 mM for EGCG, see below and reference 20). All the values collected are reported in Table 1: Kdtp (around 2 mM), and n (around 3) seems to be insensitive to the addition of sugar. It is also noteworthy that the residue-specific 1H chemical shift changes of the peptide upon EGCG addition are insensitive to the addition of sugars (supplementary Figure S5): the most responsive residues, attributed to EGCG binding sites, are P2, P9-P11, G13-G14.

Figure 2. : Variation of free sugar area (% of total signal of 5.6 mM glucose () 10 µM/1.8 mM eq galacturonic acid pectin () or 1.3 µM/ 1.8 mM eq hexose AG () vs EGCG concentration (mM)


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in a wine-like buffer. Solid lines represent the best fit obtained using eq 5, for which the KdTS were estimated to 320 mM, RMS 8×10-4 for glucose, 90 mM, RMS 6×10-9 for pectin and 20 mM, RMS 3x10-9 for AG.

Table 1. Binding data. Dissociation constant (Kdtp) and number of EGCG binding sites (n) between EGCG and IB7-14 were obtained from the fit of the experimental chemical shift variations of NH of G8, G4, K5 and G14 and Hε of K5. The number of binding sites of EGCG on the peptide does not vary significantly and is close to 3. Kdtp is a mean of values obtained from fits of the different chemical shift variations, and the RMS given are mean values of all the RMS obtained from the different fits. conditions

Kd tp (mM) n RMS

-

1.9 ±0.2

3 3×10-6

Pectin (1g/L) 1.9±0.4

3 3×10-6

AG (0.3 g/L) 1.7±0.2

3 2×10-6



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Figure 3. Chemical shift variations (∆δ) of IB7-14 (Hε K5 at 2,85 ppm) as a function of EGCG concentrations (mM). The solution contains 0.5 g/L of Arabic Gum. Symbols correspond to the measured variations and the line, to the simulation using eq. 4 until a 2.5 mM EGCG concentration 2

-7

(KdTP, 1.65 mM, ∆δmax, 0.022, χ , 6x10 ). Above 3mM of EGCG a non-specific binding occurs parameterized by a straight line.

Some interesting information can be deduced from these results: the disappearance of the 1H NMR signal of EGCG when sugars are added or that of sugars when EGCG is added could be reasonably associated with an interaction that occurs between EGCG and sugars (see below, HRMAS NMR Spectroscopy). However, the presence of sugars does not affect at all the peptide behaviour. The CMC of EGCG was estimated, and does not vary in the presence of the different sugars beyond the limits of the standard deviation (see supplementary Figure S4). It is noteworthy that the interaction inducing the disappearance of the EGCG or sugars signals depending on the titration performed, is not correlated to any apparent haze, cloud or even precipitate formation in the NMR tube during the experiments. The fraction of sugars or EGCG not


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visible in the solution state 1H NMR spectra recorded using a liquid-state NMR standard probe can be assigned to the formation of a supramolecular sugar - EGCG complex, with part of the signal escaping detection accounted for large chemical shift anisotropy and/or dipolar coupling, as previously reported for trans-anisole emulsion.21 In these conditions, the disappearance of the signal can be fitted using equation (6) and gives the dissociation constants between EGCG and the different sugars tested (Kdts). The values are reported on Table 2, and show that Arabic Gum is most strongly bound to EGCG.

Table 2. Dissociation constants (Kdts) between EGCG and different sugars. Values were obtained by fitting the decrease of the sugar resonances, corresponding to the remaining free sugars in solution, using equation (5). The values are expressed as real Kd ts or as equivalent hexose Kdeq ts. This last value takes into account the concentration in hexoses (the Pectin used corresponds to a polymer of 1800 glucuronic acid monomers, and the Arabic Gum to a polymer composed of 13800 hexose monomers). Sugars added

Kd ts

Kdeq ts (Hexose equivalent)

Glucose

320 mM

320 mM

Pectin

90 mM

0.5 mM

Arabic Gum

20 mM

13 µM

DOSY NMR spectroscopy. DOSY NMR experiments were recorded for each sample to follow the size of the complex formed. In the absence of IB7-14, the diffusion coefficient of EGCG evolves differently when sugars are added to the mixture. Fitting the experimental data using eq (4) with A=D gives rise to some interesting parameters and, notably, the maximal variation of D (Dmicelle,) which corresponds to the Diffusion coefficient of EGCG embedded in the micelle, and consequently to the size of the complex. Surprisingly, Dmicelle is higher for the mixture containing sugars (up to 2.4×10-10 m2.s-1) than for EGCG alone (1.8×10-10 m2.s-1) as displayed in figure 4 and Table 3. The hydrodynamic



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radius (RH) of EGCG deduced from Dmicelle, using the Stokes-Einstein relationship (5) is around 10 Å without any added sugar (corresponding to a micelle composed of around 6 EGCG molecules) and decreases to 7 Å in the presence of 5.6 mM glucose, 10 µM pectin or even 1.3 µM of AG (corresponding to a micelle composed of about 2 EGCG molecules). This result gives interesting information on the association process between sugars and EGCG: the decrease of the size of the micelles formed when sugars are added, which is in accordance with the formation of a EGCGsugar supramolecular complex that escapes liquid state NMR detection, suggests that sugars prefer to associate with EGCG micelles instead of free molecules. Competition takes place between the two association processes: on the one hand, the self-association of EGCG and on the other hand, the association between EGCG and sugars as suggested by the tendency of Katt to decrease from 45 M1

, without sugars, to 35 M-1, in the presence of 1.3 µM AG, without impacting the EGCG CMC in a

significant way (Table 3).

Table 3. Influence of sugars on self-association constant (Katt), Diffusion coefficient in the micelle (Dmicelle), micelle Hydrodynamic radius (RHmicelle) and Critical Micelle Concentration (CMC) values of EGCG. Katt, Dmicelle were estimated by fitting the experimental Dobs values obtained at different [EGCG] using equation 2 (where A=D) with Dfree 3.2 ±0.2×10-10 m2s-1. Values reported are those obtained with the lowest RMS value. RHmicelle is calculated from the StokesEinstein equation. CMC are calculated from the same data as explained in ref 5. EGCG Katt (M-1) Dmicelle (10-10 m2.s1 ) RH micelle (Å) RMS (10-3) CMC (mM)

45 1.8

EGCG+ 5.6 mM Glucose 40 2.35

EGCG + 10uM pectin 40 2.35

EGCG + 1.3 uM AG 35 2.4

12.0 1.0 4.4±0.6

7.1 0.6 4.7±0.5

7.1 1.5 4.6±0.6

7.0 0.6 4.4±0.7


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Figure 4. Variation of the diffusion coefficient (D) vs EGCG concentration without any sugars (), 5.6 mM glucose (∆), 10 µM pectin () or 1.3 µM AG () . Lines correspond to the best fit using eq. (2) with Ka, Dmicelle and RMS values reported on Table 3.

In the presence of the peptide IB7-14, the evolution of D values through the EGCG titration was not sensitive to the addition of sugars as shown in figure 5 (right part), confirming that sugars do not associate with the EGCG-peptide complex. The D value of the complex EGCG-IB7-14 is close to 1.8×10-10 m2.s-1 and does not evolve when pectin (in green) or AG (in red) is added to the EGCGpeptide complex (in black). This D value corresponds to a 10 Å hydrodynamic radius independent of the presence of sugars.



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Figure 5. 1H DOSY NMR spectra (700 MHz, 298K) recorded in a wine like medium of 0.5 mM IB7-14 and 1.75 mM EGCG alone (black), with 10 µM pectin (green) or 1.3 µM AG (red).

HRMAS NMR spectroscopy

The loss of a part of the polyphenol signal (close to 25 % when AG concentration reaches 1 g/L), without any change either in the turbidity of the sample or in the development of any haze or precipitate, suggests the formation of aggregates containing EGCG of a sufficient size to generate dipole-dipole interactions and susceptibility distortions, i.e. to form aggregates of radii higher than 15 nm but lower than 1 µm since the solution still remains clear22. Since HRMAS provides the ability to use all the repertoire of high resolution NMR on soft matter23, by reducing dipolar and susceptibility of “gel-like” samples by recording spectra at a high speed rotation and magic angle, experiments were performed to regain the lost signals. 1H NMR Spectra of EGCG (10mM) were recorded in the absence or presence of AG (1 g/L), using classical liquid NMR or HRMAS (rotation rate 5 kHz, figure 6).


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Figure 6. 1H NMR spectra (600 MHz) of 10 mM EGCG alone in D2O recorded in a standard liquid-state probe (bottom), 10mM EGCG with 1 g/L AG in a standard Liquid probe (centre), and in a HR MAS probe (rotation 5kHz, top). * residual water (HDO), ** A6 and A8 protons disappeared along time due to exchange with D2O.

Table 4 reports on the areas obtained for the EGCG 1H NMR signals in the different experimental conditions. It clearly shows that adding 1 g/L of AG induces a loss of around 25 % of the EGCG signal, which was fully recovered when the spectrum was recorded at Magic Angle with a spinning rate of 5000 Hz.

Table 4. Area of EGCG signals in absence/presence of AG measured on spectra recorded in liquid state or HR MAS. * Area are means of the 2, 3, 4, 2’/6’ and 2”/6” EGCG proton resonances with respect to TSP resonance (1%) . Samples composition

Experimental conditions Area of EGCG *

EGCG 10 mM

Liquid state


100 ±1


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HR MAS (5kHz) EGCG 10mM + AG 1 g/L Liquid state HR MAS (5kHz)

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100 ±2 75 ±4 100 ±2

This experiment underlines that the signal portion of polyphenols or sugar lost in liquid-state NMR should be recovered when proton spectra were recorded by spinning at magic angle, suggesting that the signal disappearance could be related to the portion implicated in the polyphenols/sugar colloids formation. The integration measurement of the lost sugar signal (glucose, pectin or Arabic gum) during EGCG titration used above as a probe to characterise the polyphenols/sugars interaction is, thereby, justified.

Dynamic Light Scattering.

Dynamic Light Scattering (DLS), which allows particle sizing up to 1 nm, was used to assess the size evolution of the sugar-polyphenol complexes with increasing EGCG concentrations. For this purpose, Arabic Gum was chosen since a solution of 0.6 g/L gives an intensity correlation function exploitable, whose analysis provides the diffusion coefficient of AG, and consequently its mean size calculated to be close to 12 nm. Correlation functions were recorded for different solutions of 0.6 g/L Arabic Gum in which EGCG was gradually added until a final concentration of 10 mM. The signal decay, fitted with a multiple exponential function, gives the size distribution, which increases concomitantly with the concentration of EGCG (Figure 7). From an average hydrodynamic radius close to 12 nm in absence of EGCG, the size of the colloids formed reaches a radius of 20 nm for a [EGCG] up to 8 mM, suggesting that the size of the colloidal complex is too small to promote the appearance of a cloudy aspect of the solution but sufficiently big to render them unobservable in liquid state NMR.


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Figure 7. Distribution size evolution of a 0.6g/L AG solution at which EGCG is gradually added (EGCG concentration range between 0 and 10mM). Rh represents the hydrodynamic radius in nm.

Molecular Dynamics Two kinds of molecular dynamic runs were performed in which the pseudo-concentration of EGCG and glucose were changed. In the first run the 6 EGCG molecules and 6 glucose molecules (approx. 10 mM) were regularly placed in a box full of water. The behaviour of EGCG seems to be independent of the presence of glucose molecules: a first micelle composed of 3 EGCG appears at around 1.5 ns and a micelle composed of the 6 EGCG molecules is formed since 50 ns. During the trajectory, the glucose molecules seem to ignore the presence of the polyphenol micelle as suggested by the number of hydrogen bonds formed between glucose and EGCG. The estimation made for the 30 last ns of the



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trajectory was of 12 H-bonds, suggesting that too weak interactions occur at these concentrations to stabilize the complex. In the second run, 36 EGCG and 36 Glucose, corresponding to a concentration of 60 mM each, were placed in the box. As expected, the first events observed since the first ns are the aggregation of polyphenols in a micelle, whose size increases throughout the molecular dynamics calculation. Glucose molecules are progressively adsorbed by the tannin micelles, to form a big object composed of all the molecules since 30 ns. A molecular rearrangement of the aggregate occurs with time in which glucose molecules surround the polyphenol micelles (Figure 8, supplementary Figure S6a). 102 H-bonds were counted in the last 30 ns of the trajectory, for which the glucose molecules, through their OH function, appear systematically as the H-donor. Meanwhile, the EGCG molecules play the role of H-acceptor, principally through the carboxyl function of the galloyl part (supplementary, Figure S6b). At the end of the dynamic calculation, the radius of gyration of the complex was evaluated to 15 Å. It is noteworthy that the radius of gyration does not take into account the solvation/hydration of the complex, and thus is intrinsically lower than the hydrodynamic radius that can be obtained by DOSY NMR experiments.


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Figure 8. Molecular Dynamics. Final snapshot showing the Glucose (in blue) embedding the EGCG molecules (in red).

DISCUSSION : This study focused on describing the techniques necessary to understand the association network of a ternary mixture composed of elements with specific behaviour. To illustrate the adequacy of NMR methods to decipher such a complex network, we studied the influence of polysaccharides like Arabic Gum and pectin on the interaction with the polyphenols-protein complex responsible for the astringency perception

24

that may be charted despite their size, molecular intricacy and low

solubility in aqueous solution. In this study, the polysaccharides used were chosen due to their already known role in the taste of wine: Arabic Gum is currently added to wine at the end of the winemaking process to sweeten the astringency sensation25 and pectin which is a degraded product of flesh grape cell walls is known to be correlated to a reduction in astringency26. Concerning glucose, it has been used to represent the



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total amount of fermentable carbohydrates which reaches a concentration close to 1g/L in dry wine 27

and as the constitutive element of more complex oligosaccharides. For this purpose, the following different approaches were used: Quantitative liquid state 1H NMR

experiments show the disappearance of part of the signal of sugars and EGCG for the different titrations performed which was not correlated to the formation of any haze, precipitate or cloud. In these conditions, the intensity of the peptide signals was not affected at all. The recovery of the totality of the lost signals was achieved by using HR-MAS NMR, suggesting that EGCG and sugars were not able to tumble sufficiently rapidly, causing a large broadening of their resonances responsible for their non-detection using standard liquid NMR. DLS experiments were also performed in order to follow the size of colloids formed when EGCG was progressively added to AG: the average colloid size increases up from 12 nm to an object of 20 nm radius, in accordance with an association process between the two entities. The size of the colloid formed is too small to make the solution cloudy, but sufficiently high to escape liquid NMR detection. To confirm this association between EGCG and sugars, molecular dynamic calculations, performed in a box containing both partners, supports the formation of a colloidal complex formed by glucose and EGCG and aids understanding of the nature of this interaction: hydrogen bonds stabilized the complex in which sugars act as donors and EGCG as acceptors. Finally, DOSY NMR experiments, that give access to the size of the supramolecular complexes, which are detectable using standard liquid NMR, provide information about the preference of sugars to complex polyphenols in their colloidal state. Even if such a colloidal complex formation was already described using different approaches, and different models of polysaccharide/polyphenols12,

28, 29

, the contribution of the present work

specifies the molecular details of such a molecular recognition, and some physical parameters characterizing the formation of this non-covalent and reversible supramolecular assembly. KdST values between EGCG and the different sugars were estimated using the disappearance of signals as


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a probe to follow the association phenomenon: AG is the more related sugar (Kdts of 20 mM), ahead of pectin (around 90 mM) and glucose (around 300 mM). Kdtp of EGCG with respect to the peptide seems insensitive to the addition of the three kinds of sugars used in this study and reaches a value close to 2 mM. This is due to the difference of affinity of EGCG for the peptide and sugars: EGCG is 10 to 150 more related to IB7-14. In these conditions, the part of EGCG bond by the sugars is negligible, that limits the competition mechanism with the saliva peptide.

CONCLUSION. The main point of the present work is summarized in Figure 9. The central role belongs to polyphenols that are in equilibrium between two states depending on their CMC. These states determine the nature of their interaction with saliva Proteins (as previously demonstrated) via a specific interaction below their CMC, this interaction becomes non-specific when above and generates the formation of hazes and/or precipitates. The presence of sugars will affect this equilibrium by sequestering part of colloidal polyphenols to form an object than can reach a 20 nm radius depending on size of the polysaccharide. This size is sufficient to prevent detection by liquid-state NMR, but not too large to cause a visible haze. This sequestration directly impacts the active concentration of polyphenols susceptible to interact with saliva proteins.



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9Å

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a

>μm

d

Kdtp= 2mM 8Å

a

Kdst= 20 to 300mM**

-1

Katt= 35 to 45 M * 5Å

a

b

15 Å to Kdttt= 22 to 28 mM

20 nm 12 Å

c

a

Figure 9. Proposed binding model in which the interaction between polyphenols and PRP peptides depends on the colloidal state of the polyphenols as previously described (Cala et al, Langmuir 2012, ref 4) and taking into account the role of sugars. * depending on the presence of sugars (see table 3) ; ** depending of the nature of sugar (see table 2). Radii were obtained from DOSY NMR experiments (a), molecular dynamics (b), dynamic light scattering (c) or estimated from the formation of precipitate (d).

ASSOCIATED CONTENT Supporting Information. Supporting Information Available, containing supplementary figures and tables indicated in the text free of charge via the internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected] AUTHOR CONTRIBUTIONS The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript that corresponds to part of PhD work of Benoit Faurie


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ACKNOWLEDGMENT The authors want to thank the Conseil Interprofessionnel du Vin de Bordeaux (CIVB, 1 cours du XXX Juillet, F-33075 Bordeaux, France) for financial support, the Région Aquitaine for supporting equipment of CESAMO and IECB, the Université de Bordeaux and the CNRS. I.P. gratefully acknowledges Nathan McClenaghan who assists us to improve the manuscript with respect to the English language. ABBREVIATIONS

AG: Arabic gum; EGCG: Epigalocatechin Gallate; PRP: Proline-Rich Protein

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