A complex plant cell wall polysaccharide rhamnogalacturonan II.A structure in quest of a function

A complex plant cell wall polysaccharide:rhamnogalacturonanII.A structure in quest of a functionS.Pérez a,*,M.A.Rodríguez-Carvajal a ,T.Doco baCentre de Recherches sur les Macromolécules Végétales,CNRS,associated with University Joseph-Fourier,BP 53X,38041Grenoble cedex,FrancebUnitéMixte de Recherches Sciences pour l’Œnologie,Laboratoire des Biopolymères,2Place Viala,34060Montpellier,FranceReceived 9December 2002;accepted 4February 2003AbstractWalls of growing plants are extremely complex and sophisticated composite materials incorporating a dynamic assembly of polysaccha-rides,proteins and phenolics.Among the polysaccharides,the pectins encompass a group of acidic heteropolysaccharides;they offer a repertoire of structural complexity associated with the occurrence of,at least,three specific domains.Whereas most of these domains are notable for their structural heterogeneity,one of these,the so-called rhamnogalacturonan II (RG-II)exhibits a remarkable conservation throughout the plant kingdom.RG-II is thought to be the most complex plant polysaccharide on Earth (MW 5–10kDa);its occurrence and strong conservation may indicate that it plays a major role in the structure and growth of higher plants.The present paper examines the most recent findings related to the occurrence,the structures,biosynthesis,biological role and properties,functional properties and technological applications of RG-II.Particular emphasis is given on the description of the three-dimensional structures of RG-II,in its monomeric and dimeric form as elucidated from the concerted investigations throughout 800MHz NMR spectroscopy,light scattering,atomic force microscopy along with molecular mechanics and dynamics.Some attempts of deciphering of the structural role that RG-II may play in the cell wall of growing plants are presented.©2003Éditions scientifiques et médicales Elsevier SAS and Sociétéfrançaise de biochimie et biologie moléculaire.All rights reserved.Keywords:Cell wall;Polysaccharides;Pectins;Structure conformation1.IntroductionThroughout their lifetime,the cells of living plants con-tinue to divide.These cells have the unusual property of being able to grow indefinitely and retaining the quality of young plants.Plant cell walls are distinguished from animal cells by the presence of a “wall”within which complex physicochemical and enzymatic processes take place.In the course of cell growth,the dimensions of the cell wall change according to the type of macromolecule of which it is com-posed.The first wall deposited after cell division is called the“middle lamella”and is essentially composed of pectic mate-rial [1].The cell then lays down a wall composed of pecto-cellulosic material (“primary”cell wall)to supplant the “middle lamella”.The primary cell wall is a glycol-proteinaceus layer composed of pectin,cellulose,hemicellu-lose,and proteins.During their growth,the plant cell can extend hundreds,and sometimes thousands,of times their original length.This occurs while the plants maintain its resistance to extreme tension (up to several hundreds of mega-Pascal on the rela-tively thin cell wall (0.1–1µm in cross-section))generated by the turgor pressure.Cell walls must yield to allow a cell to grow,while such a growth is accompanied by an increase in the total area of the wall that must maintain its thickness.This dual functionality of the cell walls is mainly achieved throughout the mixture of polysaccharides and proteins that control the continuous assembly and extension of cell walls.Cleavages,rearrangements and cross-linking of polysaccha-rides are some of the main events that characterise such a dynamic and complex assembly.Among these polysaccha-rides,those belonging to the pectic family form a matrix.They provide a proper environment for the deposition,slip-page and further extension of the cellulosic-glycan network.The original vision of the cell wall as a dead,structureless box has evolved into a dynamic picture,where highly com-plex structures are performing a diversity of functions into*Corresponding author.Tel.:+33-4-76-03-7603;fax:+33-4-76-54-7203.E-mail address:serge.perez@rs.fr (S.Pérez).Biochimie 85(2003)109–121/locate/biochi©2003Éditions scientifiques et médicales Elsevier SAS and Sociétéfrançaise de biochimie et biologie moléculaire.All rights reserved.DOI:10.1016/S 0300-9084(03)00053-1the life of plants.At least five physiological roles can be attributed to the polysaccharide components:(1)to provide an alternate co-extensive network,(2)to cement cells to-gether at the middle lamella,(3)to limit porosity of the cell wall,(4)to accommodate the symplast with an ionic and aquatic environment,and (5)to transduce environmental signals to cells throughout bioactive fragments.Thus,the “cell wall ”is very much an active metabolic compartment,despite being external to the plasma membrane,and can be best viewed as an “extracellular matrix ”.The term “pectins ”encompasses a group of acidic het-eropolysaccharides with distinct structural domains.They are subjected to both biosynthetic and cell wall-based modi-fications.The chemical structure of pectins has been the subject of many scienti fic investigations for decades.Similar to any other plant polysaccharides,pectins are polymolecular and polydisperse,exhibiting signi ficant heterogeneity with respect to both chemical structure and molecular weight,which is in the range of 100kDa.The composition of plant cell wall polysaccharides varies with source and conditions of extraction,location,and many other environmental fac-tors.Despite such a wide structural diversity,pectins can nevertheless bedescribed in terms of a “canonical ”structure,which is depicted in Fig.1.The pectic backbone is primarily a polymer of D-galacturonic acid (homopolymer of (1→4)-a -D-galacturonic acid)with varying degree of carboxyl groups methyl esteri fied and rhamnogalacturonan I (het-eropolymer of repeating a -L-rhamnosyl (1→2)linked to a -D-galactosyluronic acid disaccharide units).The a -D-galacturonic acid exists as a 4C 1conformer,with the C-6carboxyl group being in the equatorial position.As for the HGA moiety,it has been shown that irrespective of the conformations adopted at the glycosidic linkage,the direc-tion of the chain remains practically constant.Any local adjustment,as a response to a change,either in structure (e.g.de-esteri fication)or neighbourhood (e.g.counter-ions)is not likely to perturb the overall direction of the chain [2].Within the HGA region,a persistence length occurs for about 15monosaccharides.This is suf ficient to provide enough stabi-lisation of the junction zones that may be formed with Ca 2+,since it is known that in vitro,maximum stability of junction zones is obtained for oligomers containing around 15Gal p A units.This provides some latitude for the occurrence of interrupting esteri fied Gal p A units.The presence of (1→2)-linked L-rhamnose in a strictly alternating fashion in the main backbone only slightly reduces the extension of the polysaccharide chains and does not introduce any signi ficant structural perturbation in the chain direction.The nature of the side chains of the so-called rhamnogalacturonan I (RG-I)moiety may display a wide structural diversity,not only in terms of the constituting sugars:arabinofuranosyl,galacto-syl,but also in terms of glycosidic linkages:(1→5)-a -L-arabinofuranosyl in the case of arabinans,(1→4)-b -D -galactan in the case of type I arabinogalactans,(1→3)-b -D -galactan in the case of type I arabinogalactans,and (1→6)-b -D -galactan in the case of type II arabinogalactans,for which three-dimensional features have been proposed [3].The structure of the rhamnogalacturonan II (RG-II)moi-ety is presented in the following sections and figures.For the sake of simplicity,the schematic representation of HGA,RG-I,and RG-II,is given assuming that these three domains are covalently linked;this point is not firmly established.ItFig.1.Schematic representation of the “canonical ”primary structure of pectins.For the sake of simplicity,the schematic representation of HGA,RG-I,and RG-II is given assuming that these three domains are covalently linked,although this point is not firmly established.110S.P érez et al./Biochimie 85(2003)109–121has been shown[4]that HGA and RG-II both have a polyga-lacturonic acid backbone.In the case of RG-I,no direct evidences have been found for the occurrence of such cova-lent linkages,although RG-I is solubilised from cell walls with endo-polygalacturonases.Moreover,an RG-I-like frag-ment has been detected when studying RG-II[5],which could indicate the neighbourhood of these fragments.2.RG-II—the occurrenceMost of the components of the pectic matrix are notable for their structural heterogeneity.However,treatment of pri-mary cell walls of higher plants by endo-a-1,4-polygalacturonases releases a low molecular weight(5–10 kDa)structurally complex pectic“mega-oligosaccharide”, that has been named“rhamnogalacturonan II”,abbreviated to“RG-II”[6].RG-II is solubilised by treating plant cell walls with pectinolytic enzymes,mainly endo-polygalacturonase.This enzyme acts specifically on the ho-mogalacturonan backbone of pectins.It releases such frag-ments as low molecular weight oligosaccharides,RG-I and RG-II,which are separated and purified throughout a se-quence of anion-exchange and size exclusion mercial cocktails of pectinases can be used to quantitatively release RG-II from plant materials.Interest-ingly,the structure of RG-II has been shown to be virtually the same in every plant analysed hitherto;it is present in several species belonging to the families of Brassicacea, Cucurbitaceae,Leguminoseae,Apiaceae,Liliaceae, Araceae,Amaryllidaceae,Gramineae...(Table1).Antibody-based studies have indicated that RG-II occurs widely in all primary cell walls,that it may be absent from the middle lamella region,and that there is a base-labile modification ofRG-II within the cell wall[7].3.RG-II—the extraction—the red wine trackFermented beverages such as cider and wine[8,9]can be used as a direct source of RG-II since part of the industrial process involves degradation of the fruit cell walls with pectinases produced by the fermentation microflora.The RG-II(in its monomeric(mRG-II)and dimeric(dRG-II) form)is isolated by precipitation with ethanol or ultrafiltra-tion of the wine and purified by a combination of anion-exchange and size exclusion chromatography.The monomer and dimer are separated using high-resolution size exclusion columns.The concentration of RG-II is increased by enzy-matic maceration and liquefaction during processing.There-fore,despite the fact that RG-II accounts for less than5%of the primary cell wall,its resistance to fragmentation by all known pectinolytic enzymes explains its unusual high con-centration in products obtained by enzymatic treatment. Wines and fruit juice may have,depending on the source, content ranging between50and400mg/l of dRG-II.Wine and primary cell wall RG-II have identical structures [8].RG-II is present in wine as a monomer and a dimer that is cross-linked by a1:2borate-diol ester[10,11].The RG-II dimer being present in red wine at pH between3.5and3.8 indicates that the di-ester cross-linking is somehow stable between pH3and4.The fact that RG-II can be readily isolated from red wine in quantities opened the route to structural determination throughout high-resolution NMR in solution.4.RG-II—the different levels of structural organisation 4.1.The primary structure—the monomerCurrent knowledge of the primary structure of RG-II is largely based on two approaches:(1)glycosyl linkage com-position(determined by methylation analysis),and(2)iden-tification of various oligosaccharide fragments with mass spectrometry(obtained by mild acid and/or enzymatic hy-drolysis of RG-II followed by chromatographic purification). RG-II is best described as a homogalacturonan backbone of roughly seven to nine residues bearingfive oligosaccharide side chains,A-E(Fig.2)[12].Some of the backbone galac-turonic acid residues are methyl-esterified and the B-chain aceric acid(B3)and2-O-Me-Fuc p residues(B4’)are acety-Table1Origin of RG-IIOrigin Common name Reference Acer pseudoplatanus Sycamore[6]Allium cepa Onion[42]–Pectinol AC(commercialenzymolitic preparation)[43] Solanum lycopersicum Tomato[44] Glycine max Soybean[44] Pseudotsuga menziesii Douglasfir[45] Angelica acutiloba Kitagawa Japanese angelica[46] Actinidia deliciosa Kiwifruit[47] Oryza sativa Rice[48]–Red wine[8] Blupeurum falcatum Blupeurum[33] Glycyrrhiza uralensis Asian liquorice[33] Ramonda myconi Pyrenean primrose[49] Phyllostachys edulis Moso bamboo[50] Arabidopsis thaliana Mouse-ear cress[51] Cryptomeria japonica Japanese cedar[52]Beta vulgaris L.Beet[53] Pisum sativum Pea[11] Raphanus sativus Radish[10] Panax ginseng C.A.Meyer Ginseng[54] Malus domestica Apple[9] Daucus carota Carrot[9] Nicotiana tabacum L.Tobacco[55] Solanum tuberosum L.Potato tuber[56] Citrus lemonia Lemon[57]Pinus densiflora Japanese red pine[58] Chenopodium album mb’s-quarters[24] Phoenix canariensis Canary Island date palm[59] Rhopalostylis sapida Nikau palm[59] Atractylodes lancea DC.Atractylodes[60] Cucurbia moschata Duchesne Pumpkin[4]111S.Pérez et al./Biochimie85(2003)109–121lated[13,14].Recently,Vidal et al.[15]have published aconvincing NMR investigation of an oligosaccharide frag-ment of RG-II from red wine that contained a hexasaccharide main chain bearing the B and D side chains(Fig.3).Most of the proton assignments were reported in that study and it has been demonstrated that the original configurations estab-lished for the anomeric centres of two residues in the B-chain,B3and B4,were incorrect.Moreover,they showed that the B-and D-side chains were located at thefifth(M B= M5)and sixth(M D=M6)a-Gal p A residue from the reducing end,respectively.The Kdo-containing disaccharide C-side chain was detected in non-stoichiometric amounts in this oligosaccharide fragment and,consequently,it was removed to increase homogeneity and facilitate analysis;thus,the exact branch point of the C-chain is still unknown,although it must be located in this part of the molecule.Finally,some heterogeneity has been reported for RG-II from various sources,including further substitution by rhamnosyl(3-linked to B5)[9,16]and arabinosyl sugars(3-linked to the galacturonan backbone[17])as well as incomplete substitu-tion by several of the terminal sugars(A5,B5,and B7).This heterogeneity could arise from partial degradation during extraction and purification steps.Thefirst molecular mechanics study[18]of the four oligosaccharide side chains of RG-II explored the stable orientations of all the glycosidic linkages of the side chain sugars as well as the puckering preferences of the apiosyl and aceric acidfive-membered rings.The preferred conformers were partially validated by the sequential nuclear Overhauser effects(NOEs)described in thefirst NMR investigation[19] of the intact saponified and borohydride-reduced RG-II monomer,mRG-II-ol.This latter study placed the A-side chain at the penultimate a-Gal p A residue from the non-reducing end and revealed some long range contacts.How-ever,spectral assignments relied heavily on the available primary structure which has been modified since[15].4.2.Attempts to derive the3D structure by high-resolution NMR and molecular modellingThese events prompted us:(1)to assign as much of the 800MHz proton spectrum as possible,with a view to acquire a better quality proton-carbon chemical shift correlation data,(2)to evaluate the orientations of the glycosidic link-ages of both the side-and main-chain residues,(3)to confirm the primary structure determined using other physicochemi-cal methods,(4)to establish the branch points of the side chains where possible,and(5)to obtain long range interchain NOE data for validating3D models in future molecular modelling studies.The collection of new13C-NMR acquired at800MHz allowed the assignment of most of the signals corresponding to the primary structure[5].Unfortunately,some assign-ments were still ambiguous and several of the spin systems, that have been clearly identified,have never been demon-strated in previous studies of RG-II.The study has revealedFig.2.Schematic representation of the primary structure of RG-II monomer(mRG-II).Since itsfirst identification in1978by Darvill et al.[6],the primary structure of RG-II has been studied mainly by methylation analysis and mass spectroscopy of the oligosaccharides from its partial degradation.Thus,side chains (A-D)as well as a main chain of polygalacturonic acid similar to that found in HGA have been identified and characterised[12,37].However,the location of the linkage of these side chains to the main backbone has remained uncertain for over two decades.An initial NMR study of complete RG-II[19]gave two possible distributions that were in agreement with NMR.A previous work of Vidal et al.[15]showed the location of B-and D-chains,but also a change in the anomeric configuration of two of the residues of the B-chain;thisfinding made a reassignment of NMR data necessary.The proposed distribution for primary structure [5],based on new NMR assignments,is presented.Hopefully,this work will terminate the total“sequencing”of the most complex plant polysaccharide structure.112S.Pérez et al./Biochimie85(2003)109–121the presence of an RG-I backbone repeating unit in mRG-II from red wine.The determination of the glycosylation site of this fragment would shed light on the relative locations of the RG-I and RG-II component in the pectic network.In spite of this,a large body of NMR spectral data has been collected,especially concerning the assigned NOEs,and it has been applied to the preferred conformation of mRG-II in solution.The conformational analysis used the previously reported adiabatic energy surfaces along with the locations of thelocal energy minima for all disaccharide components of RG-II [18](Table 2).These data,along with the NOEs,were combined to determine possible time-average conformations of monomer mRG-II in solution.Two families of conformers,stemming from the two pos-sible diastereospeci fic assignments of one hydrogen (H3b(B1))were obtained.Both of them are in agreement with other experimental data,like the general shape of the mol-ecule and the accessibility of apiose in A-chain.When calcu-lated distances were compared with the NOEs,good agree-ment was obtained,at least qualitatively in most cases;the intensity of the expected NOEs deviated from the experimen-tal ones only for very few interactions.Considering,on the one hand,that the NOEs correspond to a time-averaged or virtual structure,and on the other,that the isolated-spin approximation had been applied to systems with nearby protons,we considered the agreement to be very good.The study has been an iterative process between the com-putational study and the NMR assignments.Thus,initial conformations for RG-II or its fragments led to the revision of known signals and the assignment of new ones that,inturn,resulted in a new set of NOEs constraints or the modi-fication of the previous ones.The resulting structures yielded slightly longer distances after minimisation than expected in the case of most long range NOEs.Nevertheless,the opti-mised conformers could be considered as good starting ge-ometries for further studies on the molecular dynamics of theFig.3.Structure of the “limit-pectin ”oligosaccharide [15],along with a depiction of the 3D structure of one of the lowest energy conformations and the chromatographic pro file of its separation after enzymatic fragmentation of mRG-II.The pro file corresponds to a size-exclusion chromatography of saponi fied and NaBH 4-reduced mRG-II (mRG-II-ol,thick line)and the products resulting from its enzymatic fragmentation (thin line).Conformation was calculated by applying a systematic search approach (view Fig.4).Table 2Disaccharidic fragments in RG-II DisaccharideUnitsb -D-A p i f -(1→2)-a -D-Gal p A A1→M A ,B1→M B b -L-RhaI-(1→3')-b -D-Apif A2→A1,B2→B1a -D-Gal p A-(1→2)-b -L-Rha p A2'→A2b -D-Gal p A-(1→3)-b -L-Rha p A2″→A2a -L-Fuc p -(1→4)-b -L-Rha pA3→A22-O -Me-a -D-Xyl p -(1→3)-a -L-Fuc p A3'→A3b -D-Glc p A-(1→4)-a -L-Fuc p A4→A3a -D-Gal p -(1→2)-b -D-Glc p A A5→A4a -L-Ace f A-(1→3)-b -L-Rha pB3→B22-O -Me-a -L-Fuc p -(1→2)-b -D-Gal p B4'→B3b -D-Gal p -(1→2)-a -L-Ace f A B4→B3a -L-Rha p -(1→3)-a -L-Ara p B5'→B4a -L-Ara p -(1→4)-b -D-Gal p B5→B4a -L-Rha p -(1→2)-a -L-Ara p B6→B4b -L-Ara f -(1→2)-a -L-Rha p B7→B6a -Kdo p -(2→3)-a -D-Gal p A C1→M C a -L-Rha p -(1→5)-a -Kdo p C2→C1b -D-Dha p -(2→3)-a -D-Gal p A D1→M D b -L-Ara f -(1→5)-b -D-Dha p D2→D1a -L-Ara f -(1→3)-a -D-Gal p A E1→M E a -D-Gal p A-(1→4)-a -D-Gal p AM x →M x +1113S.P érez et al./Biochimie 85(2003)109–121RG-II monomer and the conformational analysis of theRG-II dimer.Fig.4shows a 3D representation of the lowest energy conformations of RG-II monomer found.The general shape of the mRG-II resulted in a quite flat structure,with a length of about 37Åfor the main chain (horizontal diameter)and roughly the same length between the longest side chains (A and B)(vertical diameter).The general shape was a disc,with a thickness of about 17Å,which is in agreement with the results obtained by translational self-diffusion and light-scattering (vide infra).Preliminary results of mRG-II molecular dynamics are presented in Fig.5The starting geometries do not change considerably throughout the dynamics.Thus,the main chain maintains a stiff shape close to an extended helix,with a mean extension of around 38Å.This value is close to the expected one for a 9-unit pectate helix adopting a twofold (38.2Å)or threefold (39.9Å)conformation.Moreover,ga-lacturonic glycosidic torsion angles explore the minima found in molecular mechanics (φaround 60–80and W around 180–270)[20].According to these results,the pres-ence of RG-II side chains does not affect the extension and direction of HGA.This fact suggests that the interaction of galacturonic acid with calcium cations in muro could initiatethe approaching of mRG-II as a previous step to the dimeri-sation with boron.A-and B-chains are also fairly extended throughout the dynamics,especially the latter.Both adopt an orientation perpendicular to the main chain,although the structure re-mains quite planar.Glycosidic torsion angles explore regions close to the starting minima.A higher flexibility is observed in linkages involving apiosyl residues,owing to their fura-nosic nature and the fact that the linkage Rha p-(1→3’)-A p i f involves a hydroxymethyl group.4.3.The structural and hydration features of monomeric RG-IIThe translational self-diffusion coef ficient of the mRG-II has been established with the pulsed-field gradient spin-echo technique [21].When this parameter is interpreted according to the hydrodynamic theory for a simple model,its molecular volume corresponds to a radius of roughly 20Å.4.4.The 3D of dimeric RG-II by light scattering and molecular modellingLight scattering provides a direct way to assess the ten-dency for a molecule to aggregate and offers an estimate ofFig.4.A 3D representation of the lowest energy conformations of RG-II monomer (mRG-II).NOE data were applied to molecular modelling through a systematic search method,included in the Sybyl program [38].This method does not apply any kind of energy minimisation to the resulting conformers;hence strong steric interactions can arise that cause the conformation to become extremely unfavourable.In an attempt to reduce this kind of interaction,hydroxylic protons were removed,as their orientation could be easily modi fied in a further geometry optimisation.The glycosidic torsion angles were modi fied at steps of 30°according to a range of values arising from the adiabatic energy maps.All the conformations in which the distance between atoms was less than the sum of their van der Waals radii should be rejected,although a softening multiplicator factor of 0.8–0.7was applied to the van der Waals radii so that more conformations met this criterion.Finally,the distance constraints were de fined:only those conformers that provide a distance in the range of 0–5Åwere taken into account (0–6Åif distance included a methyl group),without considering the intensity of the corresponding NOE.Finally,hydroxylic protons were added and the molecules minimised.114S.P érez et al./Biochimie 85(2003)109–121the hydrodynamic dimensions of the species under study.mRG-II and dRG-II samples were submitted to this charac-terisation.The hydrodynamic radii (R h )were calculated to be 19.2±0.5Åfor the monomer and 17.8±0.3Åfor the dimer,i.e.no increase in R h is detected.These results suggest that dimerisation involves a compaction of the 3D structure of each monomeric component,accompanied by an association of their largest area.Moreover,the results match those ob-tained from translational self-diffusion (vide supra)and from NMR-molecular modelling.4.5.The NMR of dimeric dRG-IIHigh-resolution (800MHz)1H-and 13C-NMR spectra of dRG-II have been acquired.The objective is to identify as many NOE connectivities as possible to apply them in a molecular modelling study.A similarity with the NMR from monomer has been observed,but with some differences.Some of them arisefrom different preparations of samples:in the previous study [19]mRG-II had been saponi fied and reduced,whereas the dimer has not been treated in this way to be closer to the in vivo state (M.A.Rodr íguez-Carvajal et al.,in preparation).Because of this,and besides the modi fi-cations from the dimerisation with boron,signals for methyl esters and acetyls appear and modi fications of chemical shifts of protons and carbons close to these groups are ex-pected.Indeed,patterns of NOEs equivalent to those de-picted in the monomer have been found,indicating that both conformations present zones with similar orientations,at least locally.4.6.Atomic force microscopyAtomic force microscopy (AFM)studies have been per-formed on monomer mRG-II and dRG-II and the results are shown in Fig.7.The resulting pro files presented broad peaksFig.5.MD simulation of mRG-II in water solution and trajectories of the variations of some descriptors as a function of time.Molecular dynamics of structures from the systematic search were carried out by using the program AMBER 6[39]complemented with GLYCAM [40]parameters for sugars.6912molecules of water were added to the starting structure in a cubic box of 62Åon each side.Cubic boundary conditions were selected,and Particle Mesh Ewald method applied to evaluate electrostatics.A cut-off of 9Åwas applied to non-bonded interactions.Equilibration of the solvent was carried out by minimisation followed by a short (25ps)dynamics keeping the solute fixed.Then,the whole system was minimised,applying decreasing restraints to the solute molecule.Finally,the completely minimised system was heated (50ps)from 10to 300K at constant pressure and maintained at 300K for the rest of the dynamics,with a thermal bath coupling and a pressure bath coupling both of 1ps.The integration step was 2fs and all bond lengths were kept constant using the SHAKE algorithm.Data were registered for 0.4ns.On the left panel,several snapshots of dynamics,taken every 100ps,together with the 650closest molecules of water corresponding to the first and second solvation shells,are represented.On the right panel,evolutions of some descriptors of the general shape of mRG-II are presented (extension of main chain,torsion angle between A-and B-chains,and RMS).115S.P érez et al./Biochimie 85(2003)109–121。