Line Sahli

Adeline Boire

Véronique Solé-Jamault

Hélène Rogniaux

Alexandre Giuliani

Pierre Roblin

Denis Renard

New exploration of the -y-gliadin structure through it s partial hydrolysis

Whe.it storage proteins lntrinsicaUy disordered proteins 30 structure modeUing

The partial enzymatic hydrolysis ofwheat gliadins constitutes an interesting tool to unravel their structural spec ificity. In this work, the structure and conformation of -y gliadin were investigated through its limited chymotrypsic digestion. Using a combination of computational, biochemical and biophysical tools, we studied each of its N and C terminal domains. Our results reveal that -y gliadin is a partially disordered protein with an unfolded N terminal demain surprisingly resistant to chymotrypsin and a folded C terminal dornain. Using spec troscopic tools, we showed that structural transitions occured over the disordered N terminal domain for de creasing ethanol/water ratios. Using SAXS measurements, low resolution 30 structures of-y gliadin were proposed. To relate the repeated motifs of the N terminal demain of-y gliadin to its structure , engineered peptide models PQQPY/F were aise studied. Overall results demonstrated similarities between the N terminal demain and its derived model peptides. Our findings support the use of these peptides as general templates for under standing the wheat protein assembly and dynamics.

Introduction

Wheat gliadins are storage proteins that be long to the large prola mins family (1 5]. There are synthesized in the lumen of the endoplas mie reticulum (ER) and stored into micrometer sized organelles called protein bodies (PBs) (6 8].Although these proteins are known to be es sential during seed germination and growth, so far, their structure re mains hypothetical.

Gliadins are monomeric proteins, soluble in an alcohol water mix ture or aàdic solutions [ 1,9, 1 0]. According to their electrophoretic mo bility and isoelectric focusing, there are classified into three main types: cr/ �. 'Y (sulfur rich) and ro gliadins (sulfur poor) [START_REF] Shewry | The prolamin storage proteins of cereal seeds: structure and evolution[END_REF][START_REF] Popineau | Fractionation and characterisation of γ-gliadins from bread wheat[END_REF][START_REF] Wieser | Chemistry of gluten proteins[END_REF][START_REF] Larre | Fractionation of gliadins from common wheat by cation exchange FPLC[END_REF]. The amine aàd sequences of sulfur rich gliadins consist of two separate demains: a N terminal demain composed of repeated sequences, and a non repetitive C terminal demain containing disulfide bonds (3,4 ,10,13,14]. The sulfur poor gliadins sequence contains a long repet itive domain, lacking cy steines. Repetitive do mains of whole gliadins are composed by interspersed repeats of motifs rich in praline, glutamine, and tyrosine (cr/ �. ro gliadins) or phenylalanine ("/ gliadins) (13.15]. Previous spectroscopie studies have shown that their repeated demains result of poly praline II (PPII) helix and� turn structures, while their sulfur rich demains are essentially composed of O'. helix ( 16 18]. Small Angle X ray Scattering (SAXS) measurements of purified gliadins [START_REF] Thomson | Molecular images of cereal proteins by STM[END_REF][START_REF] Thomson | Small angle X-ray scattering of wheat seed-storage proteins: α-, γand ω-gliadins and the high molecular weight (HMW) subunits of glutenin[END_REF] or gliadins mixtures [START_REF] Sato | Molecular assembly of wheat gliadins into nanostructures: a smallangle X-ray scattering study of gliadins in distilled water over a wide concentration range[END_REF][START_REF] Boire | Dynamics of liquid-liquid phase separation of wheat gliadins[END_REF], in aqueous ethanol mixture [START_REF] Thomson | Small angle X-ray scattering of wheat seed-storage proteins: α-, γand ω-gliadins and the high molecular weight (HMW) subunits of glutenin[END_REF][START_REF] Boire | Dynamics of liquid-liquid phase separation of wheat gliadins[END_REF] or aàdic solutions [START_REF] Thomson | Molecular images of cereal proteins by STM[END_REF], were performed. Ali the findings converge to de scribe gliadins as extended proteins with an ellipsoidal conformation [START_REF] Urade | Gliadins from wheat grain: an overview, from primary structure to nanostructures of aggregates[END_REF]. In this present work, we in tend to reconsider the structure of glia dins in the light of advances in the field of intrisically disordered pro teins (IDPs) (24 27]. A better understanding of their structure will allow to determine the contribution of these speàfic sequences in the self assembly and storage of gliadins in wheat seed.

'Y gliadin, comprising bath a N terminal repetitive demain and a C terminal non repetitive demain, has been chosen as a representative mode! of gliadins. Due to the polymorphism of wheat proteins, various isoforms of-y gliadins have been identified (9.12]. To avoid this molec ular diversity, we only focus on the-y44 isoform.As previously described [START_REF] Legay | Comparative study of enzymatic hydrolysis of α/β and γ-gliadin[END_REF], extracted -y44 gliadin was enzymatically digested and released do mains were purified separately. To unravel the importance of the re peated motifs in the aforementioned process, pentapeptides of (PQQPF) and (PQQPY) representing the consensus sequences, respec tively found in 'Y gliadins and cr/ p/ ro gliadins, were designed. The influ ence of their length on their structure was aise studied by repeating those 8 or 17 times. In this study, we showed that 'Y gliadin is partially disordered. With SAXS measurement, we proposed a partial 30 struc ture at low resolution of-y44 gliadin and we discussed the limitations

Analytical characterization

For biochemical characterization, protein powders of γ44 gliadin and its domains (150 μg) were solubilized in Tris HCl pH 8/ethanol mix ture (60/40) (v/v) and applied on an analytical column of Nucleosil C 18 (300 Å, 5 μm, 250 × 4 mm, Macherey nagel) equilibrated with deionized water containing 0.06% of TFA. Elution was performed using an acetoni trile gradient containing 0.06% TFA (15 100%) in 60 min. Protein sam ples were diluted with Laemli buffer and heated at 95 °C for 5 min for SDS PAGE analysis (4 12% Bis Tris Plus Gels, Bolt™, Invitrogen). After migration, electrophoresis gel was incubated overnight in Instant blue solution (Expedeon) to ensure gel coloration. In the case of the N terminal domain, a further silver nitrate staining was carried out. The gel was rinsed in distilled water and scanned.

Mass spectrometry (MS) analysis

Purity and molecular weight of proteins were determined using a SYNAPT G2Si HDMS mass spectrometer (Waters, France) with an electrospray source of ionization (ESI). Measurements were performed in the positive ion mode, on a mass to charge ratio (m/z) range of 800 3000. (PQQPY) 17 was analyzed at 0.1 mg/mL in a water acetonitrile mixture (50/50) (v/v) containing 0.1% formic acid (v/v). Do mains were analyzed at 0.2 mg/mL in 60% of acetonitrile (v/v). Samples were infused at a flow rate of 5 μL/min and spectra were recorded over 2 min. Spectrum processing was done using the MassLynx™ software (Waters, France).

Dynamic light scattering (DLS)

DLS measurements were performed using a zetasizer Nano Series (Nano ZS, Malvern instrument, Germany). Protein powders were dis solved in 0.05 M Na 2 HPO 4 /NaH 2 PO 4 pH 7.2, 55% ethanol (v/v), under stirring, overnight, at room temperature. Residual undissolved material was removed by filtration on a 0.2 μm membrane filter (Sartorius, France). Protein samples (100 μL, 2.5 mg/mL) were analyzed in a quartz cuvette (Hellma analytics) with a path length of 10 mm. The measure ments were performed in manual mode: 10 runs of 60 s were collected and repeated three times for each measurement (20 °C). The correlation functions were analyzed using the CONTIN method.

Synchrotron radiation circular dichroism (SRCD)

Measurements were performed using the DISCO beamline at Soleil synchrotron (Gif sur Yvette, France) using the SRCD endstation [START_REF] Réfrégiers | DISCO synchrotron-radiation circular-dichroism endstation at SOLEIL[END_REF].

The samples were prepared in 50 mM MOPS buffer pH 7.2, 25 mM NaCl at 20% or 55% ethanol (v/v). At 55% ethanol, protein samples are concentrated at 2 mg/mL, except (PQQPY) 17 concentrated at 0.72 mg/mL. At 20% ethanol, protein samples are concentrated as fol lows: 1.5 mg/mL for N terminal domain, 1.5 mg/mL for C terminal do main, 1.5 mg/mL for mixed domains (molar ratio 1:1), 0.53 mg/mL for (PQQPY) 17 , 2 mg/mL for (PQQPY) 8 and 2 mg/mL for (PQQPF) 8 . Each spectrum is the average of three acquisitions. The spectrum of buffer was subtracted from the protein spectrum using the CDtoolX software (http://www.cdtools.cryst.bbk.ac.uk/). Spectrum was smoothed using the Savitzky Golay filtering (order 3 out of 9 points). Content of second ary structures was determined using the BestSel software (http:// bestsel.elte.hu/index.php).

Small angle X ray scattering (SAXS)

SAXS measurements were performed at the Chemical Engineering Laboratory (LGC Toulouse, France) with the XEUSS 2.0 (Xenocs Com pany) composed of X ray micro source delivering at 8 keV a spot sized beam equal to 0.5 mm with intensity close to 30 × 10 6 photons/ s. The sample to detector distance of the X rays was 1216.5 mm of this approach. We demonstrated strong similarities between the N terminal domain and the model peptides.

Material and methods

In silico analyses

The analysis of protein sequences was carried out using ExPASy (https://web.expasy.org/protscale/) and CIDER (https://pappulab. wustl.edu/CIDER/) tools. Structural predictions were performed by i Tasser software (https://zhanglab.ccmb.med.umich.edu/I TASSER/). In the case of γ gliadin and its domains, referenced and reviewed P08453 accession was used (UniprotKB).

Purification of domains

γ44 gliadin was extracted and purified from wheat gluten of cv. Hardi as previously described [START_REF] Banc | Structure and orientation changes of ωand γ-gliadins at the airwater interface: a PM-IRRAS spectroscopy and Brewster angle microscopy study[END_REF]. Domains were obtained by the chy motryptic digestion of the γ44 gliadin [START_REF] Legay | Comparative study of enzymatic hydrolysis of α/β and γ-gliadin[END_REF]. γ44 gliadin was first solubi lized (10 mg/mL) in Tris HCl pH 8/ethanol mixture (60/40). Chymotrypsin contained in 10 mM CaCl 2 buffer, was added at en zyme/protein ratio of 1: 500. Enzymatic hydrolysis was carried out at room temperature, for 24 h, under continuous stirring. Hydrolyzed sam ples were then applied on a semi preparative column of Nucleosil C 18 (300 Å, 7 μm, 250 × 10 mm? Macherey nagel) equilibrated with deion ized water containing 0.06% of trifluoroacetic acid (TFA). Elution was performed using an acetonitrile gradient containing 0.06% TFA (15 100%). The N terminal domain does not absorb at 280 nm, elution was therefore monitored at 215 and 280 nm. Purified domains were col lected. Acetonitrile and TFA solvents were evaporated using a rotatory evaporator. Fractions were then freeze dried and stored at room temperature.

Synthesis and purification of peptides

(PQQPY) 8 and (PQQPF) 8 were synthesized and purified on an Inertsil ODS 3 column (4.6 × 250 mm) by GenScript (Piscataway NJ, USA). The synthetic gene of (PQQPY) 17 was inserted on the pETb32 vec tor (Novagen) and expressed as a fusion protein with thioredoxin and His 6 tag as previously described [START_REF] Elmorjani | Synthetic Genes Specifying Periodic Polymers Modelled on the Repetitive Domain of Wheat Gliadins: Conception and Expression[END_REF][START_REF] Sourice | High microbial production and characterization of strictly periodic polymers modelled on the repetitive domain of wheat gliadins[END_REF]. Asp Pro residues were intro duced between the recombinant peptide and the thioredoxin/His tag to allow their subsequent separation. Protein expression was performed in Escherichia coli BLR (DE3) pLys strain (Novagen) in the presence of 0.5 mM isopropyl β d 1 thiogalactopyranoside (IPTG). Glycerol stocks of the transformed bacteria (stored at -80 °C) were used to inoculate the overnight Luria Bertani (LB) pre culture at 37 °C. Cells were induced at 30 °C in Terrific broth (TB) media containing 25 μg/ml chloramphen icol and 50 μg/mL ampicillin. After the expression, the cells collected by centrifugation (5000 ×g, 20 min, 4 °C) were washed and re suspended in Ni NTA binding buffer (20 mM Tris pH 8, 0.5 M NaCl, and 20 mM im idazole). Cells were lysed by sonication (3 cycles of 1.5 min, 4 °C) and harvested at 20,000 ×g for 20 min. Pellets were further resuspended with the Ni NTA binding buffer supplemented with 6 M of urea. Cell de bris were removed by a final centrifugation step performed at 20,000g for 20 min. Resulting supernatants were purified by Ni NTA column (HisTrap™HP). Proteins were eluted from the column using an imidaz ole gradient (20 500 mM). Collected fractions were dialyzed against deionized water and freeze dried. An acid cleavage was carried out to recover the repetitive polypeptide. The fusion proteins were dispersed in 70% formic acid at 37 °C for 72H. The resulting samples were then loaded on the Nucleosil C 18 column (300 Å, 7 μm, 250 × 10 mm, Macherey nagel) equilibrated with 0.06% of TFA in deionized water. Elu tion was performed using an acetonitrile gradient containing 0.06% TFA (10 75%) in 90 min. Collected fractions were then dialyzed against de ionized water, freeze dried and stored at room temperature.

the weakly charged nature of the γ gliadin and its domains. The low charge content of γ gliadin and its truncated domains suggests that they belong to the weak polyelectrolytes/polyampholytes category which could adopt globules or tadpoles conformations (compact con formation) [START_REF] Das | Relating sequence encoded information to form and function of intrinsically disordered proteins[END_REF]. Similar results were recently obtained for α gliadin and low molecular weight glutenin [START_REF] Markgren | Glutenin and gliadin, a piece in the puzzle of their structural properties in the cell described through Monte Carlo simulations[END_REF]. Besides, the composition anal ysis of γ gliadin amino acid sequence suggests a significant enrichment in proline and glutamine as compared to other IDPs (see Supplementary Fig. S1). Both γ gliadin and C terminal domain are basic proteins with a theoretical isoelectric point (pI) ranging from 8 to 8.5. The N terminal domain has a theoretical pI close to 5. According to the hydropathy values, C terminal is suggested to be the most hydrophobic domain of γ gliadin. Resulting to its low complexity sequence, N terminal domain is predicted to be disordered (Table 1). Disorder predictors also suggest a disordered N terminal domain [START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF]. Further analyses through the ter tiary and secondary structural predictor i Tasser [START_REF] Roy | I-TASSER: a unified platform for automated protein structure and function prediction[END_REF] confirm these ob servations. The repetitive sequence is predicted to be unfolded while the C terminal domain is predicted to contain α helix structures. As the C terminal of the γ gliadin is predicted to be structured, a particular attention is paid to this protein domain (Fig. 1A). i Tasser simulations generate a C terminal model with a moderate confidence (C score = -1.79) (Fig. 1B). Based on these results, structural alignment program has been performed to match the C terminal model to all structures in the PDB library. According to i Tasser, the protein from the PDB that have the closest structural similarity with the C terminal domain is a globular protein, termed as Brazil nut 2S albumin (2LVF). Sequence ho mology between the non repetitive domain of α gliadin with 2S stor age proteins was already highlighted and was suggested to be related to a common ancestral gene [START_REF] Kreis | Molecular evolution of the seed storage proteins of barley, rye and wheat[END_REF][START_REF] Kreis | Unusual features of cereal seed protein structure and evolution[END_REF].

Finally, in silico results suggest that γ gliadin is a weakly charged protein, comprising two distinct domains of equivalent length that ex hibit different physical chemical properties. Analyses converge to pre dict that γ gliadin is an hybrid protein which could adopt globular or tadpole conformations. The non repetitive C terminal domain is pre dicted to be globular while the repetitive N terminal domain is pre dicted to be disordered. These predictions are in agreement with previous SRCD results [START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF]. A high content of disordered structures (46.9%) has been determined in the γ44 gliadin full length and have been attributed to its repetitive N terminal domain. However, even if the low sequence complexity of the N terminal domain is consistent with its hypothetic disorder, its low charge content contrast with IDPs [START_REF] Romero | Sequence complexity of disordered protein[END_REF]. Moreover, the absence of enzymatic hydrolysis within this domain, as previously reported by Legay et al. [START_REF] Legay | Comparative study of enzymatic hydrolysis of α/β and γ-gliadin[END_REF], raises some questions. Lim ited proteolysis experiments are usually used to probe the unfolded conformation of IDPs [START_REF] Fontana | Probing protein structure by limited proteolysis[END_REF]. The N terminal domain should therefore ex hibit hypersensitivity to proteases [START_REF] Tompa | Intrinsically unstructured proteins[END_REF][START_REF] Fontana | Probing protein structure by limited proteolysis[END_REF][START_REF] Receveur-Bréhot | Assessing protein disorder and induced folding[END_REF]. Ultimately, all these ele ments show the importance of experimentally studying the structure of each of the domains.

Composition of purified domain samples

As previously stated, γ44 gliadin has been extracted and purified from the wheat seed [START_REF] Banc | Structure and orientation changes of ωand γ-gliadins at the airwater interface: a PM-IRRAS spectroscopy and Brewster angle microscopy study[END_REF][START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF]. According to Legay et al. method [START_REF] Legay | Comparative study of enzymatic hydrolysis of α/β and γ-gliadin[END_REF], re petitive (N terminal) and non repetitive (C terminal) domains were Sequence properties of proteins. pI, isoelectric point; k, charge patterning parameter. k values close to zero indicate that oppositely charged residues are well-mixed, while k values close to one indicate that oppositely charged residues are segregated. FCR, fraction of charged residues. NCPR, net charge per residue which is the difference between the fractions of positively charged and negatively charged residues. Disorder promoting scores above 0.5 predict disordered sequences. Disorder promoting score below 0.5 predict ordered sequence. To compare with our proteins, two model proteins were also analyzed: calpastatin corresponds to a fully disordered protein model while lysozyme corresponds to a globular protein model. providing a range of scattering vector starting from 0.005 Å -1 to 0.5 Å -1 . Protein samples were solubilized in 50 mM MOPS pH 7.2, 25 mM NaCl and 55% ethanol (v/v), under stirring, overnight, at room temperature. Residual undissolved material was removed by centrifu gation (30 min, 15,000 ×g). Protein concentrations are as follows: 8.3 mg/mL for γ44 gliadin, 8.4 mg/mL for N terminal domain, 5.0 mg/mL for C terminal domain and 9.3 mg/mL for mixed domains (molar ratio 1:1). The samples were exposed for 7200 s to the beam and the scattered intensity was collected on the 1 M Pilatus detector.

Protein

The protein solvent was recorded and subtracted from the protein spec tra. Data integration, reduction and averaging were performed using the FOXTROT software.

The theoretical R g (Å) for globular protein was calculated by the fol lowing equation [START_REF] Wilkins | Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques[END_REF]: R g =(3/5) 1/2 R h ,with R h, the experimental hydro dynamic radius of the native protein.

The theoretical R g (Å) for a disordered protein was determined by the Flory's equation [START_REF] Flory | Principles of polymer chemistry[END_REF]: R g =R 0 N ν with N, the number of residues, R 0 = 2.54 ± 0.01 Å which is a constant that depends on the persistence length and ν a value comprised between ½ and 3/5. Under conditions where polymer polymer interactions are equal to polymer solvent and solvent solvent interactions, the polymer behaves as a Gaussian chain and the exponent ν is predicted to be ½. An average value ν = 0.55 was chosen corresponding to a theta solvent for the protein [START_REF] Markgren | Glutenin and gliadin, a piece in the puzzle of their structural properties in the cell described through Monte Carlo simulations[END_REF].

2.9. SAXS fitting using SasView v. 5.0 SAXS fitting using SasView v. 5.0 (https://www.sasview.org/) was used to fit experimental SAXS curves of N ter, C ter domains and γ44 gliadin by geometrical models.

2.10.Ab initiomodelling

The collected data of the entire protein was analyzed using the ATSAS program package (https://www.embl hamburg.de/biosaxs/ atsas online/) and in particular the GASBOR program which consists in the ab initio reconstruction of a protein structure by a chain like en semble of dummy residues and the MONSA program where a low reso lution beads model is proposed using two phases and the three SAXS curves.

Results and discussion

Sequence properties and structural predictions of the γ gliadin domains

To determine the main features of the primary structure of γ gliadin domains, in silico analyses were performed. The P08453 accession (Uniprot KB) of γ gliadin is used for this purpose. Note that previous work has shown the high similarity between sequences of all the refer enced γ gliadins found in the Uniprot KB database [START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF]. The amino acid sequences were run through CIDER [START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF] and ExPASy tools to be com pared with a fully disordered protein (calpastatin P20810) and a globu lar protein (lysozyme, P83673). The fractions of charged residues (FCR) and the net charge per residue (NCPR) presented in Table 1 highlight [START_REF] Shewry | What is gluten-why is it special?[END_REF][START_REF] Shewry | The prolamin storage proteins of cereal seeds: structure and evolution[END_REF], where a C-score of a higher value signifies a mode! with a higher confidenoe.

obtained from the limited hydrolysis of-y44 gliadin and detailed in the Supplementary Information. To determine the composition of purified domain samples, analytical chromatography and mass spectrometry analyses were performed. Purified N terminal domain is eluted be tween 31% and 34% of acetonitrile (Fig. 2B, /e/t). Elution is monitored at 215 nm because the N terminal does not absorb at 280 nm (no tryp tophan residue ). Probably due to the speàfiàty of its sequence and its Jack of basic amino acids, the N terminal domain is not revealed by the Coomassie staining. N terminal domain is only visible after sil ver ni trate staining of the gel (-16 17 kDa). Mass spectrometry experiments determined a molecular mass of 14.4 kDa ( Fig. 2B, right) with a mass ac curacy of about 50 ppm. The existence oftwo populations in the protein sample is also highlighted (ô = 3. 8 kDa), however, the 14.4 kDa form appears to be largely predominant Purifie<! C terminal domain also dis plays two populations, with the major one at 16.6 kDa (mass accuracy of about50 ppm) (Fig. 2C, right, speàes A). Sorne heterogeneity is observ able (speàes A' and A" on Fig. 2C) as well as a second population, with Jess abundance (B and B' species, at 17.5 kDa). This heterogeneity is also observable by SDS PAGE and could be explained by the non speàfic enzymatic hydrolysis (Fig. 2B, /eft). As described in the Supple mentary Information, it appears that the C terminal domain is slightly cleaved by the chymotrypsin digestion. This results in the release of two peptides ( 14 kDa and 3 kDa) under reduàng conditions (Fig. S1 ). Contrary to the C terminal domain, N terminal domain exhibit over estimated apparent molecular weight (SDS PAGE, Fig. 2A&B). This elec trophoretic profile was also observe<! in the case of the -y44 gliadin full length [START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF] and suggests a conformational speàfiàties of the domain, in line with our previous predictions. Since disordered proteins are known for their abnormal electrophoretic mobility due to their hydrodynamic radius, higher than those of the globular protein ( such as C terminal do main) [START_REF] Pitt-Rivers | Ambesi Impiombato, The binding of sodium dodecyl sulphate to various proteins[END_REF][START_REF] Iakoucheva | Aberrant mobility phenomena of the DNA repair protein XPA[END_REF], this may suggest that the repetitive N terminal is indeed disordered.

Finally, all purifie<! samples show a satisfactory protein content and homogeneity. Isolated domains exhibit Jow protein heterogeneity de spite the non specific hydrolysis they have undergone. As previously re ported [START_REF] Legay | Comparative study of enzymatic hydrolysis of α/β and γ-gliadin[END_REF], the chymotrypsin, which preferentially cleaves upstream of aromatic amino acids, does not cleave within the N terminal domain, even after 24 h of incubation while it is a phenylalanine rich domain.

The o/igomeric state of )'44 gliadin and its purified do mains in good solvent

The hydrodynamic properties of-y44 gliadin and its domains under good solvent conditions (55% ethanoljwater mixture, pH 7.2) were ex plored by Dynamic Light Scattering (D15) . Autocorrelation functions, showed in Fig. 3 , argue to the homogeneity of protein solutions. Appar ent hydrodynamic radius (RgPP) of 5.1 nm (-y 44 gliadin) (Fig. 3A), 4.6 nm (N terminal domain) (Fig. 3 B) , 3.0 nm (C terminal domain) (Fig. 3C) and 5.0 nm (mixed domains at molar ratio of 1 :1) (Fig. 3D) were determined. The N terminal domain radius is equivalent to the whole protein radius. These results may emphasize the expanded con formation of the N terminal domain contrasting with the compact con formation of the C terminal domain. As aforementioned, disordered proteins have Jarger hydrodynamic dimensions compare<! to conven tional globular proteins [START_REF] Wilkins | Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques[END_REF][START_REF] Pitt-Rivers | Ambesi Impiombato, The binding of sodium dodecyl sulphate to various proteins[END_REF]. RgPP of mi xed domains is close to the -y44 gliadin full length, however, as the two domains have similar mo Jecular weights, it is difficult to differentiate them by D15. It cannot be therefore concluded that the two domains restore the entire protein (Fig. 3D ). The evolution of RW"' proteins in presence of increasing ionic strength was also investigated. The presence of 15 mM NaO does not af fect the RgPP or the solubility of proteins (Fig. 3 ), while the addition of 50 or 100 mM Jeads to protein aggregation (noisy correlograms).

A/ N-terminal domain

The -y44 gliadin intrinsic disorder arises from its N terminal do main

To estima te the secondary structure content of the -y gliadin do mains, synchrotron radiation circular dichroism spectra in far ultraviolet region were recorded in good solvent conditions (0.05 M MOPS pH 72, 25 mM NaO and 55%ethanol (vjv)) (Fig. 4). The spectro scopie study of the full length -y44 gliadin was previously reported [START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF]. Spectral deconvolution ofN terminal domain spectrum using Bestsel software [START_REF] Micsonai | BeStSel: a web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra[END_REF] suggests the high disordered content of the N terminal domain (51.5% unordered structures, 2.5% O'. helix, 27.3% (l, strand, 18.7% turn). Surprisingly, the C terminal domain would also exhibit a high disorder content ( 41.8% unordered structures, 32.6% O'. helix, 9.4% (l, strand, 16.3% turn). On the other hand, the high O'. helical secondary structure proportion found in the C terminal moiety is highlighted. Sig na! of the mixed domains spectrum is greatiy noisy (Fig. 4A). The N and C terminal spectra were averaged to construct a theoretical mixed do mains spectrum and submitted to the Bestsel software. The predictions obtained (23.2% O'. helix, 16.3% (l, strand, 15.8% tum and 44.3% unor dered structures) show no noticeable differences with the secondary structure content of the-y44 gliadin full length (Fig. 4B).

Finally, spectroscopie data suggests that N terminal is a Jow struc tured domain and C terminal is a domain rich in a helix. These observa tions are in line with the previous in silico analysis. These results are also in agreement with the established structural data of the -y gliadins [START_REF] Tatham | The conformation of wheat gluten proteins. The secondary structures and thermal stabilities of α-, β-, y-and ω-gliadins[END_REF][START_REF] Tatham | Conformational studies of peptides derived by the enzymic hydrolysis of a gamma-type gliadin[END_REF], but it should be noticed that the Bestsel tool does not allow to quantify polyproline II type helix, considering them as disordered struc tures. Conceming the high disorder content found in the C terminal do main, this is consistent with the high content of coi! predicted by i Tasser (Fig. 1A). However, this may also be related to the partial hydrolysis of the C terminal domain as shown in the Supplementary ln formation. The deconvolution of CD spectra into structural elements rel evant for IDPs relies on datasets including denatured proteins [START_REF] Sreerama | Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis[END_REF]. The interpretation in terms of intrinsic disorder should therefore be taken with care.

N terminal domain appears to fold upon decreasing ethanol content

The evolution of secondary structures of proteins at 20% of ethanol was also investigated ( 0.05 M MOPS pH 7 2, 25 mM N aa and 20% eth a no! (v/v)) (Fig. 5). As-y44 gliadin undergoes a phase separation under these conditions [START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF], SR CD has not been measured on the full length protein. Sorne structural transitions are observed when the water con tent of samples is increased (Fig. 5 & Table 2). N terminal domain tends to Jose disordered structures in favor of(l, strands ( from 51.5% un ordered structures, 27.3% (l, strands at 55% ethanol (v/v) to 41.1% unor dered structures, 38.9% (l, strands at 20% ethanol (v/v) ). C terminal domain folds into O'. helix (from 32.6% at 55% ethanol (v/v) to 45.2% at 20% ethanol (v/v) ), while disordered content slightiy increases (from 41.8% at 55% ethanol (v/v) to 49.1% at 20% ethanol (v/v)). A Joss in (l, structures ( strands, tums) is observable and would explain the increase of a helix and disorder contents. Mixed domains still have a high disor dered content at 20% ethanol (v/v) (15.9% O'. helix, 14.9% (l, strand, 15.1% tum and 54.1% unordered structures). Since data of mixed do mains at 55% ethanol were obtained by theoretical approach, they could not be compared with our present results. seems to gain ex helix and disordered content in detriment to the r, structures. The presence of disorder to order transitions within the N terminal may be consistent with the propensity of IDPs to this type of transition, due their flexibility and Jack of structural constraint [START_REF] Tompa | Intrinsically unstructured proteins[END_REF][START_REF] Uversky | Natively unfolded proteins: a point where biology waits for physics[END_REF][START_REF] Berlow | Functional advantages of dynamic protein disorder[END_REF]. its derived domains are displayed in Fig. 6A where the absolute scattered intensity I ( cm-1 ) is plotted versus the scattering wave vector Log q (A-1 ). The Guinier analyses (for qR g < 1.0) give a radius of gyra tion (Rg) of 62.5 A for -y44 gliadin, 39.6 A for N terminal domain and 27.1 A for C terminal domain (Supplementary Information). The -y44 gliadin Rg value is much higher than those previously determined in the literature (26.6 A in 1% acid acetic and 38 A in 70% ethanol (vfv)) [START_REF] Thomson | Molecular images of cereal proteins by STM[END_REF][START_REF] Thomson | Small angle X-ray scattering of wheat seed-storage proteins: α-, γand ω-gliadins and the high molecular weight (HMW) subunits of glutenin[END_REF]. However, R g is strongly dependent on solvent conditions. Jt ranged from 43.8 to 69.7 A by decreasing water content in ethanol/ water mixtures [START_REF] Orecchioni | Gliadin characterization by sans and gliadin nanoparticle growth modelization[END_REF]. To deepen our analyses, we use the previously de termined R h values to calculate the theoretical Rg values. According to a globular mode! [START_REF] Wilkins | Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques[END_REF], a theoretical Rg of232 A was determined for the C terminal domain. Considering the protein disorder, a Rg of 612 A was calculated for the -y44 gliadin using Flory's equation [START_REF] Flory | Principles of polymer chemistry[END_REF] (Material and methods). The same equation was used to establish a theoretical Rg of 41.7 A for the N terminal domain. On the other hand, the relation dedicated to fully unfolded protein models [START_REF] Wilkins | Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques[END_REF] shows a considerable overestimation of the R g of the N terminal domain (120 A). Finally, the experimental Rg values are in very close agreement with theoretical ones, meaning that the conformations and solvent conditions were fully taken into account in the equations used. M:eP of 51 kDa (-y44 gliadin), 22 kDa (N terminal domain) and 41 kDa (C terminal domain) were estimated from the extrapolated scattering intensity at zero angle 1(0) (Fig. 6A). In comparison with the data derived from mass spectrometry, it clearly appears that molec ular weights extrapolated from SAXS experiments are Jargely overestimated. This result may be justified by the presence of aggre gates in the samples and the slight upturn of scattering intensity at Jow q values. The disorder of the N terminal domain and the order of the C terminal domain are visible in the shape of the pair distance In the case of the full length -y44 gliadin and the N terminal domain, Krakty plots displayed in Fig. 6C show the presence of a plateau from 0.1 A-1 , in agreement with their high disorder content ("unfolded" conformation). An increasing slope at high q values of -1.7 is obtained for the N ter domain, value characteristic of unstructured proteins. While a slope of -4.0 is found for the C ter domain, value characteristic of a globular shape. The presence of a maximum at 0.08 A-1 in the C terminal curve confirrns the compact or globular con formation of this domain (Fig. 6B). Finally, a slope of -2.0 is found for the -y44 gliadin, value intermediate between those of the N ter and the C ter domains. Scattering data of the N terminal domain was fitted on the 0.005 0.5 A-1 scattering vector q range by a flexible cylinder mode! ( or wormlike chain mode!) with a length of 392.9 A a Kuhn length of 21.3 A and a radius of 3.5 A (x 2 = 2.9) while the C terminal domain was fitted by a tri axial ellipsoid mode!, with a minor, major and polar radii of 11.1, 22.9 and 54.6 A respectively (x 2 = 2.4) (Supplementary Information). In addition, the fi tting of the full length protein on the 0.01 0.5 A-1 scattering vector q range from a combination of the two form factors identified for the two domains, Le. sum of tri axial ellipsoid plus wormlike chain models, gives a very reasonable result (x2 = 2.8).

Tablez Structural comparison at 55%and 20%of ethanol (v/v). Oifference values (Oiff) correspond to the dilferences of the values obtained at 55%and 20%ofethanol (v/v).

Ethanol % N-terrninal domain C-terrninal domain

However, as the cross term between the two form factors is neglected in the fitting procedure, the final result has no physical meaning and is considered as being not full y satisfying.

Low resolution ab initio models of-y44 gliadin

To determine the overall shape of -y44 gliadin, Jow resolution models were computed using an ab initio approach. GASBOR software [START_REF] Svergun | Determination of domain structure of proteins from x-ray solution scattering[END_REF] was run using the reciprocal space mode (slow calculation). The symmetry of the protein was left as P1 (no symmetry, default value). The proposed model is shown in Fig. 6D. The γ44 gliadin is predicted to be an elongated protein with an estimated length of 195 Å (Fig. 6D,a). Similar conformation was obtained using the DAMMIF software [START_REF] Franke | DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering[END_REF] (data not shown). A strong structural similarity with the globular Brazil Nut 2S albumin (2LVF) has been previously suggested by i Tasser predictions. To confirm these predictions, the scattering profile of the Brazil Nut 2S albumin was calculated using CRYSOL [START_REF] Svergun | CRYSOL -a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates[END_REF] and com pared (through its PDB structure) to the experimental scattering data of the C terminal domain. No good fitting was obtained between the Brazil Nut 2S albumin theoretical curve and the C terminal experimental curve (Supplementary Information). The same observation was done when the C terminal domain scattering data was compared with the i Tasser model. These results suggest structural differences between the domain and the two models predicted by the i Tasser tool.

For comparison, γ44 gliadin was submitted to MONSA software [START_REF] Svergun | Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing[END_REF]. This multiphase dummy atom modelling tool allows the simulta neous fitting of multiple curves leading to ab initio shape determination of a full length protein from the two domains. The proposed model of γ44 gliadin is consistent with the previous results by highlighting the worm like chain conformation of the N terminal domain (length of Fig. 6. SAXS analysis of γ44-gliadin and its domains. A. Scattering intensity Log I(q) vs Log q (A 1 ) of γ44-gliadin at 8.3 mg/mL (blue), N-terminal domain at 8.4 mg/mL (orange) and Cterminal domain at 5 mg/mL (grey) in 0.05 M MOPS pH 7.2, 25 mM NaCl and 55% ethanol (v/v). B. Pair distance distribution function p(r)/P max vs the distance r (Å) calculated from the scattering curves using the PRIMUS software [START_REF] Konarev | PRIMUS: a Windows PC-based system for small-angle scattering data analysis[END_REF]. C. Krakty plots I(q)*q 2 vs q (A 1 ) of scattering curves displayed in A. D. Low resolution model of γ44-gliadin displayed by GASBOR (a) and MONSA (b) software. a According to Flory's equation [START_REF] Flory | Principles of polymer chemistry[END_REF].

b According to a globular model equation [START_REF] Wilkins | Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques[END_REF].

154 Å) and the compactness of the C terminal domain (length of 74 Å) (Fig. 6D,b). Even if all the findings are consistent, it should be noted that ab initio calculations correspond to low resolution models that cannot necessarily reflect the reality. The absence of crystallographic data and cryo TEM observations on gliadins in solution do not allow the confir mation of these models. Finally, SAXS experiments provide ensemble averaged structural in formation about the γ44 gliadin and its isolated domains. All structural data obtained are summarized in Table 3. SAXS experiments are com plementary to CD results. They also suggest that the N terminal domain is unordered in contrast to the globular like shape of the C terminal do main. In agreement with the previous in silico analysis, the low resolution models suggest that γ44 gliadin could adopt a tadpole con formation with an ellipsoidal C terminal head. Note that the low resolution γ44 gliadin model proposed in this study is in agreement with the first γ gliadin computer molecular model proposed in 1995 [START_REF] D'ovidio | Nucleotide sequence of a gamma-type glutenin gene from a durum wheat: correlation with a gamma-type glutenin subunit from the same biotype[END_REF]. It is also consistent with a recent molecular model of a α gliadin obtained by Monte Carlo simulations [START_REF] Markgren | Glutenin and gliadin, a piece in the puzzle of their structural properties in the cell described through Monte Carlo simulations[END_REF]. In both models, an elongated shape was obtained with a tail in the vicinity of its N terminal domain. Interestingly, in the latter model, a ring like motif was also observed on snapshots when disulfide bonds were present. Similarly, a ring was ob tained on the low resolution model of the C terminal domain obtained by GASBOR (Supplementary Information, Fig. S4).

More generally, it should be pointed out that the chimeric conforma tion of the protein, extended and globular mixed conformation, has underlined the limitations of the SAXS data processing tools. Due to its hybrid nature, it is very challenging to elucidate its 3D structure by tak ing into account its two domains. MONSA software was therefore the best compromise tool to probe the conformation of the entire protein from the two domains.

In this work, we carried out a detailed structural characterization of a γ gliadin. We proved its semi disordered nature by studying the secondary and tertiary structure of each of its domains. These investiga tions were possible through a partial protein hydrolysis. The disordered N terminal domain is not hydrolyzed even after 24H of chymotrypsic digestion, its high flexibility being thought to promote the proteolytic targeting. On the other hand, the ordered protein domain is slightly hy drolyzed as shown in the Supplementary Information. All these ele ments could suggest that cleavage sites contained in the N terminal sequence of the γ44 gliadin are not accessible to the chymotrypsin, in the proteolysis conditions used (40% ethanol (v/v)). To investigate the secondary structure of γ44 gliadin in these specific conditions, further SRCD experiments were performed (Fig. 7). The resulted secondary structures content show no difference between 55% (19.4% α helix, 18.6% β strand, 15.2% turn and 46.9% unordered structures) and 40% of ethanol (v/v) (20.6% α helix, 19.2% β strand, 15.6% turn and 44.7% unordered structures). The secondary structure of the protein cannot therefore explain the absence of cleavage within the N terminal do main. Further SAXS experiments should be performed to determine the conformation adopted by the protein at 40% of ethanol. The three dimensional structure of the protein could limit the proteolysis reaction. Note that the chymotryptic insensitivity of the N terminal domain could also be related to its high proline content. Previous studies have re ported that proline turns could induce conformational hardening of proteins [START_REF] Kirkland | Evaluation of the proline-rich antigen of Coccidioides immitis as a vaccine candidate in mice[END_REF][START_REF] Fischer | Conformational specificity of chymotrypsin toward proline-containing substrates[END_REF]. It could be therefore concluded that the gaining of do mains by partial proteolysis of the entire protein has indirectly provided valuable information about the γ44 gliadin conformation.

Peptides models summarize the physicochemical properties and sec ondary structure of the N terminal domain

To determine whether (PQQPY) 8 , (PQQPF) 8 and (PQQPY) 17 behave as the N terminal domain, we carried out biochemical (Supplementary Information) and structural assays. Their primary structures were first analyzed in silico through CIDER [START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF] and ExPASy tools (Table 4). Re peated peptides exhibit a theoretical pl close to that of the N terminal domain (-5 6). As expected, (PQQPY)s, (PQQPF)s and (PQQPY) 11 are not charged peptides suggesting that they belong to the weak polyelec trolytes/polyampholytes category. As for the N terminal domain, their redundant sequences are predicted to be disordered. Regarding these predictions, peptides appear to share common physicochemical fea tures with the N terminal domain.

The secondary structure content of mode! peptides were also re corded (0.05 M MOPS pH 7.2, 25 mM NaCI) at 55% and 20% ethanol (v/v) to be compared to the other -y44 gliadin truncated forms (Fig. 8). At 55% ethanol, (PQQPF) 8 (53.6%), (PQQPY) 8 (46.9%) and (PQQPY) 17 (49.4%) display a high disorder content. The different lengths do not influence the peptide secondary structures. Note that the recombinant (PQQPY) 17 was only recorded from 190 nm. probably due to the contaminants present as aforementioned that strongly absorbed an.< 190 nm. Since (PQQPY)s and (PQQPY)11 spectra are too noisy at 20% of ethanol, only (PQQPF)s spectrum has been analyzed. In comparison to the previousSRCD results (55%ethanol (v/v) ), spectral deconvolution of (PQQPF) 8 at 20% ethanol (v/v) shows a remarkable structuration in� strand ( +9.7% of� strand and -5.6% of unordered structures). This induced folding into � strand structures at Jower eth anol content was also observed in the case of the N terminal domain (Table 2).

Taken together, all these data demonstrate that peptides exhibit structural and behavioral similarities with the repetitive -y44 gliadin N terminal domain. Other investigations on these simplifie<! sequences could enable to understand the aforementioned chymotrypsic insensi tivity of the domain. As an instance, mutation of the peptide prolines would confirm their potential role in the hypothetical conformational hardening of the N terminal. More broadly, our engineered peptides, in spired by the repetitive sequences of all gliadins, may actas mimicking models to study the self assembly of wheat proteins in further experi ments. It is still unclear how the intrinsic disorder of the N terminal do main contributes to the biological function of wheat gliadins. It may contribute to the tendency of gliadins to undergo liquid liquid phase separation highlighted in previous studies [START_REF] Boire | Dynamics of liquid-liquid phase separation of wheat gliadins[END_REF][START_REF] Sahli | Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation[END_REF]. Also, it may enhance specific interactions with the membrane of the endoplasmic reticulum during their synthesis in wheat seed [START_REF] Banc | Exploring the interactions of gliadins with model membranes: effect of confined geometry and interfaces[END_REF]. Engineered peptides mimick ing N terminal domain may help to better understand the driving force of gliadins self assembly occurring during the phases of accumulation and storage in wheat seed. A (nm)

.... •• • •••••• • •• ..

Condusion

Through partial hydrolysis and combination of bioinformatics, phys icochemical and biophysical tools, we were able to revisit the structure of a wheat gliadin mode!. We demonstrated the semi disordered con formation of the -y44 gliadin by analyzing each of its domains. Struc tural assessments underlined the difficulty to eluàdate the three dimensional structure of a hybrid protein. An approach combining SAXS curves fitting using geometrical models and ab initio computa tions revealed that-y44 gliadin could be ascribed to an elongated pro tein with a tadpole conformation and an estimated maximum distance of 200 250 A The enzymatic reaction, exhibiting a disordered N terminal resistant do main, highlighted the particularity of the studied protein and contributed to unravel its molecular features.

The structural characterization of repetitive polymers like architec ture has shown their strong similarities with the repeated gliadin se quences from which they derive. Our findings support their use as general templates for understanding the wheat protein assembly and dynamics. two major peaks corresponding to the N and C terminal domains, respectively eluted at 33% and 45% of acetonitrile (0.06% TFA) (Figure S2B). Note that the N-terminal domain, mainly composed of phenylalanins, do not absorb at 280 nm (Figure S2B, grey curve).

Guinier analyses of SAXS experiments

To determine the structure of γ44-gliadin and its domains, SAXS experiments were performed. The Guinier plots of γ44-gliadin and its derived domains are displayed in Figure S3 where Ln I (cm -1 ) is plotted vs q 2 (Å -2 ). The Guinier analyses (for qRg < 1.0) gave a radius of gyration (Rg) of 62.5 Å for γ44gliadin, 39.6 Å for N-terminal domain and 27.1 Å for C-terminal domain.

Figure S3: Guinier analyses. Guinier plots Ln I(q) vs q 2 (A -2 ) of γ44-gliadin at 8.3 mg/mL (black), N-terminal domain at 8.4 mg/mL (red) and C-terminal domain at 5 mg/mL (green) in 0.05 M MOPS pH 7.2, 25 mM NaCl and 55% ethanol (v/v). Dashed lines stand for the Guinier fit. Fitting SAXS curves using geometrical models SAXS curves of N-ter and C-ter domains were preliminary fitted using SasView v. 5.0 in order to find the best realistic geometrical models. C-ter domain was best fitted using a triaxial ellipsoid model while N-ter domain was best fitted using a wormlike chain or flexible cylinder model (Figure S4, A &B). Fitting curves of γ44-gliadin, N-ter and C-ter domains from MONSA computing were also displayed (Figure S4, C), given some confidence to the ab initio modelling.

Ab initio model of the C-terminal domain obtained using GASBOR tool

To determine the overall shape of the C-terminal domain, a low-resolution model was computed using an ab initio approach similarly to the γ44-gliadin. GASBOR software was run using the reciprocal space mode (slow calculation). The symmetry of the proteins was left as P1 (no symmetry, default value). The proposed models are shown in

  -Tasser predictions of the C-terminal domain.A. Predicted secondarystructure. H,a-het ix; C,coil. B. C-terminal model withC-score = 1.79. C-score is typicallyin the range of

5 -Fig. 2 .

 52 Fig. 2. Composition and purity of protein samples. A (Left) Chromatographie profile of N-terminal domain at 215 (black) and 200 nm (grey), eluted with aoetonitrile gradient (15-100%) containing 0.06%TFA SOS.PAGE ofN-terrninal domain (black arrow at-16-17 kOa) reve.l!ed by silver nitrate staining. The sme.ir observed at 50-110 kOa isdue to a bad destaining of the gel. (Rigltt) ESI-MS spectrum of N-terrninal domain analyzed at 02 mg/ml in 60% of aoetonitrile ( v/v). B. (Left) Chromatographie profile of C-terrninal domain at 215 (black) and 280 nm (grey), eluted with acetonitrile gradient (15-100%) containing O.D6%TFA SOS-PAGE of C-terminal domain (black arrow at-16-17 kDa) reve.l!ed by Instant blue staining. (Rigltt) ESI-MS spectrum of C-t errninal domain analyzed at 02 mg/ml in 60% of acetonitrile ( v/v).

Fig. 3 .

 3 Fig. 3. Correlograms of-y44-gtiadin and its dornains obtained by OIS.A. -y44-gtiadin. 8. N-terminal domain. C. (-terminal domain. O. Mixed dornains at molar ratio ofl :1. Correlograms are obtained at2 mg/ml in 0.05 M MOPS pH 72, 25 mM Naa and 55%ethanol (v/v) (20 •q.

3 . 6 .Fig. 4 .

 364 Fig. 4. Far-UV CO spectra of constructions under good solvent conditions. A. CO spectra of-y44-gliadin at2 mg/ml (blad<), N-terrninal domain at 2 mg/ ml (wine), (-terminal dornain at 2 mg/ml (green) and mixed dornains (1:1 molar ratio) at 2mg/ml (purple) in 0.05 M MOPS pH 7 2, 25 mM NaCI and 55%ethanol (v/v). 8. The histogramsshow secondary structures content obtained from the deconvolution of the spectra using 8estSel software.

Fig. 5 .

 5 Fig. 5.Far-UV CD spectra of constructions upon increasin gaqueous content.A CD spectra of N-terminal domain at 1.5 mg/mL(wine), (-terminal domain at 1.5 mg/rnL(green) and mixed dornains (1 :1 rnolar ratio) at 15 mg/rnL(purple) in 0.05 M MOPS pH 7 2,25 mM Naa and 20% ethanol (v/v). 8. The histograms showsecondarystructures content frorn the deconvolution of the spectra using Best.sel software.

  distribution functions p(r) (Fig. 6B). The maximum distance (D max ) found for -y44 gliadin, N terminal domain and C terminal domain are respectively of 255 A 150 A and 100 A The large Dmax of the N terminal domain suggests an extended conformation, while the Jower Dmax of the C terminal domain suggests a compact conformation. The sum of Dmax of N ter and C ter domains is in agreement with the Dmax calculated for the entire protein. The R g values calculated from the P (r) functions are respectively of 65.3 A for -y44 gliadin, 41.7 A for N terminal domain and 27.7 A for C terminal domain. P(r) functions displayed R g values closer to the experimental and theoretical R g values, in line with the Jesser sensitivity of aggregation in the R g calculation from P(r) function.

Fig. 7 .

 7 Fig. 7. Far-UV CD spectra of γ44-gliadin in 55% and 40% ethanol (v/v). A. CD spectra of γ44-gliadin in 55% (black) and 40% ethanol (v/v) (grey) at 2 mg/mL in 0.05 M MOPS pH 7.2, 25 mM NaCl. B. The histograms show secondary structures content from the deconvolution of the spectra using BestSel software.

Fig. 8 .

 8 Fig.8. Far-LN CD spectraofmode l peptides.A CDspectraof ( PQQP Y),,at0.72 mg/ml (dark blue), (PQQPY )s at2 mg/mL(l ig htblue)and (PQQPF)s at 2 m g/ ml(omnge) in 0. 05 M MOPS pH 7 2, 25 mM NaO and 55% ethanol (v/v). The spectra of (PQQPF)s at2 mg/ml (orange) in 20% ethanol (v/v) is represented in orange dotted lines. B. The histogr ams show secondary structures con tent from th e deconvolution of the spectra using BestSel software.

Figure S2 :

 S2 Figure S2: Hydrolysis of γ44-gliadin by chymotrypsin reaction. A. Enzymatic kinetics of chymotrypsin followed by SDS-PAGE 10% under non reduced (left) and reduced (10 mM dithiothreitol) (right) conditions. B. Chromatographic profile of the γ44-gliadin hydrolysates, injected on a semi-preparative column of Nucleosil C-18, observed at 215 (black) and 280 nm (grey), eluted with acetonitrile gradient (0-100% -0.06% TFA).

Figure S4 :

 S4 Figure S4: SAXS fitting. Geometrical models computed using SasView (A, B) for the N-ter (red) and Cter (green) domains of γ44-gliadin and using MONSA (C, D) for the N-ter (red) and C-ter (green) domains of γ44-gliadin and γ44-gliadin (black). Data are plotted as Intensity vs q (A, C) and as Krakty plot (B,D).

Figure S5 .

 S5 The C-terminal domain adopts a compact ellipsoidal conformation with an estimated length of 85 Å. The dimensions obtained are very similar to the one obtained by computer simulation on the amino acid sequence of a γ-type glutenin, closed to γ-gliadin's one.

Figure S5 .

 S5 Figure S5. Low resolution model of the C-terminal domain of γ44-gliadin displayed by GASBOR.

Figure S6 :

 S6 Figure S6: Comparison of the calculated scattering intensity of Brazil Nut 2S albumin and i-Tasser model to the C-terminal domain using CRYSOL software.

17 (

 17 PQQPY)8 and (PQQPF)8 peptides were chemically synthesized while (PQQPY)17 was obtained by heterologous expression30 . As with gliadin and its domain, analytical chromatography and spectrometry analyses were performed to determine the composition of peptide samples. The recombinant (PQQPY)17 is eluted at ~33% of acetonitrile and exhibits a very unusual electrophoretic migration (smear), presumably due to its very highly redundant sequence (FigureS7, left). Mass spectrometry data display a molecular weight of 10.4 kDa (FigureS7, right). The protein content of the sample is estimated to be greater than 70%. Despite the presence of non-protein contaminants, which most probably correspond to salts and/or urea used during the purification, (PQQPY)17 sample has an excellent protein purity (~100%). As aforementioned, sequences repeated 8 times were provided and analyzed by Genscript. (PQQPY)8 and (PQQPF)8 were respectively eluted at ~38% and ~47% of acetonitrile with an estimated purity of 88.5% and 95.6% (information provided by Genscipt).

Figure S7 :

 S7 Figure S7: Composition and purity of the (PQQPY)17 sample. (Left) Chromatographic profile of (PQQPY)17 at 215 (black) and 280 nm (grey), eluted with acetonitrile gradient (10-75%) containing 0.06% TFA. SDS-PAGE of (PQQPY)17 (black arrow at ~3.5 kDa) revealed by Instant blue staining. (Right) ESI-MS spectrum of (PQQPY)17 at 0.1 mg/mL in water-acetonitrile mixture (50/50) (v/v) containing 0.1% formic acid (v/v)

Table 1

 1

Mixed domains 55% 20% Oilf 55% 20% Oilf 55% 20% Oilf a-Hetix 2.5 0.7 1.8 32.6 452 12.6 232 15.9 7.3 �-Strand 27.3 38.9 11.6 9.4 3.1 6.3 16.3 14.9 1.4 Turn 18.7 19.4 0.7 16.3 2.6 13.7 15.8 15.1 0.7 Others 51

.5 41.1 10.4 41.8 49.1 7.3 44.3 54.1 9.8

Table 3

 3 Structural parameters of γ44-gliadin and its domains derived from DLS and SAXS experiments.

	Structural and hydrodynamic parameters	γ44-gliadin	N-ter domain	C-ter domain
	Molecular weight (kDa) (from mass spectrometry)	38.6	14.4	16.6
	Molecular weight (kDa) (from Guinier)	51	22	41
	R h app (Å) (from DLS)	51	46	30
	I(0) (cm 1 )	0.29	0.12	0.13
	R g (Å) (from Guinier)	62.5	39.6	27.1
	Rg Rh (from Guinier)	1.22	0.86	0.90
	R g (Å) (from P (r))	65.3	41.7	27.7
	Rg Rh (from P(r))	1.28	0.91	0.92
	R g (Å) theoretical (calculated)	61.2 a	41.7 a	23.2 b
	D max (Å)	255	150	100
	Form factor	Sum of wormlike chain and triaxial ellipsoid	Wormlike chain	Triaxial ellipsoid

Table 4

 4 Sequence properties of model peptides. pI, isoelectric point; k, charge patterning parameter. k values close to zero indicate that oppositely charged residues are well-mixed, while k values close to one indicate that oppositely charged residues are segregated. FCR, fraction of charged residues. NCPR, net charge per residue which is the difference between the fractions of positively charged and negatively charged residues. Disorder promoting scores above 0.5 predict disordered sequences. Disorder promoting score below 0.5 predict ordered sequence.

	Protein	Residues	pI	Hydropathy	k	FCR	NCPR	Proline fraction	Disorder promoting
	(PQQPY) 17	85	5.88	2.20	-	0	0	0.40	0.80
	(PQQPY) 8	40	5.92	2.20	-	0	0	0.40	0.80
	(PQQPF) 8	40	5.96	3.02	-	0	0	0.40	0.80

Vacic V., Uversky V.N., Dunker A.K., and Lonardi S. "Composition Profiler: A tool for discovery and visualization of amino acid composition differences". BMCBioinformatics. 8:211. (2007)

CRediT authorship contribution statement

Une Sahli: methodology, acquisition of data, analysis and interpreta tion of data, drafting the manuscript.

Adeline Boire: critical reading of the manuscript, supervision ofline Sahli's work.

Véronique Solé Jamault: purification of wheat proteins. Hélène Rogniaux: mass spectrometry data acquisiiton and analysis. Alexandre Giuliani: supervision at SOLEIL synchrotron beamline for SRCD experiments.

Pierre Roblin: SAXS data analysis. Denis Renard: critical reading of the manuscript, supervision of Une Sahli's work

Acknowledgments

This work was carried out with the finanàal support of the regional programme "Food for Tomorrow/Cap Aliment, Research, Education and Innovation in Pays de la Loire", whichis supporte<! by the Fr ench Region Pays de la Loire and the European Regional Development Fund ( FEDER ).

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2020.09.136.

Supplementary information

Compositional profile

The fasta sequence of γ44-gliadin was analyzed through the webserver Composition Profiler (http://www.cprofiler.org/ 1 ) to detect bias in its amino acid composition as compared to a set of proteins that display a well-defined 3D structure (PDB select 25). The analysis reveals that γ44-gliadin is significantly enriched in proline and glutamine (Figure S1A). Also, it is depleted in charged amino acids (D,E, K, R). For comparison, the protein database Disprot is compared to the PDB Select 25 database (Figure S1B). It shows that Disprot proteins are enriched in disorder promoting amino acids (in red) and depleted in order promoting amino acids (in blue). Therefore, γ44-gliadin may be a specific case of partially disordered proteins.

Hydrolysis and purification of γ44-gliadin domains

To isolate N and C terminal domains of the γ44-gliadin, chymotryptic digestion of the protein was monitored in Tris-HCl pH 8/ethanol mixture (60/40), at room temperature, during 24H. The enzymatic kinetic was followed by polyacrylamide gel electrophoresis (SDS-PAGE 10%) under reducing and non-reducing conditions (Figure S1) as previously described 28 . The appearance of two bands with a very close molecular weight (~17 kDa) is observed after 1H hydrolysis. Samples after reduction revealed that observed bands correspond to the C-terminal domain, which is known to contain all cysteine residues of the γ-gliadin 3,13 . These observations are consistent with the previous mass spectrometry data that identified the presence of two C-terminal populations (16.6 kDa and 17.5 kDa). The bands observed at 3 kDa and 14 kDa appearing after reduction correspond to fragments of the C-terminal domain. These findings show a partial hydrolysis of the C-terminal domain caused by the chymotrypsic digestion and maintained by the disulfide bonds. For unclear reasons, but certainly due to the particularity of its sequence (no basic amino acids) 44 , N-terminal domain is not revealed by Coomassie staining, and therefore not visible by SDS-PAGE (῀14kDa) (Figure S2A). The injection of the γ44-gliadin hydrolysates on C-18 column reveals the presence of