University of Manchester
Ring Puckering Landscapes of Glycosaminoglycan-related Monosaccharides from Molecular Dynamics SimulationsIrfan Alibay?,? and Richard A. Bryce?*? Division of Pharmacy and Optometry, School of Health Sciences, University of Manchester, Oxford Road, M13 9PL, UK? Structural Bioinformatics and Computational Biochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UKCorresponding Author*Richard Bryce, Division of Pharmacy and Optometry, School of Health Sciences, University of Manchester, Manchester, M13 9PT, U.K. Email: R.A.Bryce@manchester.ac.uk, Tel: (0)161-275-8345, Fax: (0)161-275-2481; ORCID 0000-0002-8145-2345AbstractThe conformational flexibility of the glycosaminoglycans (GAGs) are known to be key in their binding and biological function, for example in regulating coagulation and cell growth. In this work, we employ enhanced sampling molecular dynamics simulations to probe the ring conformations of GAG-related monosaccharides, including a range of acetylated and sulfated GAG residues. We first perform unbiased MD simulations of glucose anomers and the epimers glucoronate and iduronate. These calculations indicate that in some cases, an excess of 15 ?s are required for adequate sampling of ring pucker due to the high energy barriers between states. However, by applying our recently developed msesMD simulation method (multidimensional swarm enhanced sampling molecular dynamics), we were able to quantitatively and rapidly reproduce these ring pucker landscapes. From msesMD simulations, the puckering free energy profiles were then compared for fifteen further monosaccharides related to GAGs; this includes to our knowledge the first simulation study of sulfation effects on ?-GalNAc ring puckering. For the force field employed, we find that in general the calculated pucker free energy profiles for sulfated sugars were similar to the corresponding unsulfated profiles. This accords with recent experimental studies suggesting that variation in ring pucker of sulfated GAG residues is primarily dictated by interactions with surrounding residues rather than by intrinsic conformational preference. As an exception to this, however, we predict that 4-O-sulfation of ?-GalNAc leads to reduced ring rigidity, with a significant lowering in energy of the 1C4 ring conformation; this observation may have implications for understanding the structural basis of the biological function of ?-GalNAc-containing glycosaminoglycans such as dermatan sulfate.1. IntroductionCarbohydrates are ubiquitous biopolymers that fulfil a wide range of key functions in nature, from energy sources and structural units to mediators of biomolecular recognition. ADDIN REFMGR.CITE <Refman><Cite><Author>Dwek</Author><Year>1996</Year><RecNum>981</RecNum><IDText>Glycobiology: Toward Understanding the Function of Sugars</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>981</Ref_ID><Title_Primary>Glycobiology:<f name="Symbol"> </f>Toward Understanding the Function of Sugars</Title_Primary><Authors_Primary>Dwek,Raymond A.</Authors_Primary><Date_Primary>1996/1/1</Date_Primary><Reprint>Not in File</Reprint><Start_Page>683</Start_Page><End_Page>720</End_Page><Periodical>Chem.Rev.</Periodical><Volume>96</Volume><Issue>2</Issue><Web_URL> name="System">Chemical Reviews</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Chem.Rev.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>1 Carbohydrates are linear or branched polymers, comprised of monosaccharide residues connected by glycosidic linkages. These monosaccharide units are typically hexopyranoses, ie. six-membered rings decorated by a variety of pendant functional groups, including hydroxyl, hydroxymethyl, carboxylate, aminoacyl and sulfate groups (Figure 1a). For example, glycosaminoglycans (GAGs) are heteropolysaccharides that often include sulfated residues, forming negatively charged polymers that interact in a specific way with the basic residues of receptor proteins linked to cell adhesion and profileration. ADDIN REFMGR.CITE <Refman><Cite><Author>Jackson</Author><Year>1991</Year><RecNum>1154</RecNum><IDText>Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1154</Ref_ID><Title_Primary>Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes</Title_Primary><Authors_Primary>Jackson,Richard L.</Authors_Primary><Authors_Primary>Busch,Steven J.</Authors_Primary><Authors_Primary>Cardin,Alan D.</Authors_Primary><Date_Primary>1991</Date_Primary><Keywords>Property</Keywords><Keywords>PROTEIN</Keywords><Reprint>Not in File</Reprint><Start_Page>481</Start_Page><End_Page>539</End_Page><Periodical>Physiolog.Rev.</Periodical><Volume>71</Volume><Issue>2</Issue><ZZ_JournalStdAbbrev><f name="System">Physiolog.Rev.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Bishop</Author><Year>2007</Year><RecNum>1155</RecNum><IDText>Heparan sulphate proteoglycans fine-tune mammalian physiology</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1155</Ref_ID><Title_Primary>Heparan sulphate proteoglycans fine-tune mammalian physiology</Title_Primary><Authors_Primary>Bishop,Joseph R.</Authors_Primary><Authors_Primary>Schuksz,Manuela</Authors_Primary><Authors_Primary>Esko,Jeffrey D.</Authors_Primary><Date_Primary>2007</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1030</Start_Page><Periodical>Nature</Periodical><Volume>446</Volume><Issue>7139</Issue><ZZ_JournalFull><f name="System">Nature</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>2,3,PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkdhbWE8L0F1dGhvcj48WWVhcj4yMDA2PC9ZZWFyPjxSZWNO
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ADDIN EN.CITE.DATA 4,5 In addition to the tuning of carbohydrate conformation and interactions by glycosidic linkage and functionalisation of the pyranose ring, another important degree of freedom in these biopolymers is the conformation of the ring itself: hexopyranose rings can adopt different shapes, called puckers, including most often chair (C) forms but less frequently boat (B), half-chair (H), skew-boat (S) and other forms (Figure 1b). The various puckers can be succinctly described on the puckering conformation hypersurface by two angles, θ and ???according to the scheme of Cremer-Pople (Figure 1b). ADDIN REFMGR.CITE <Refman><Cite><Author>Cremer</Author><Year>1975</Year><RecNum>1134</RecNum><IDText>General definition of ring puckering coordinates</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1134</Ref_ID><Title_Primary>General definition of ring puckering coordinates</Title_Primary><Authors_Primary>Cremer,D.</Authors_Primary><Authors_Primary>Pople,J.A.</Authors_Primary><Date_Primary>1975/3/1</Date_Primary><Keywords>puckering</Keywords><Keywords>RING</Keywords><Reprint>Not in File</Reprint><Start_Page>1354</Start_Page><End_Page>1358</End_Page><Periodical>J.Am.Chem.Soc.</Periodical><Volume>97</Volume><Issue>6</Issue><Web_URL> name="System">J.Am.Chem.Soc.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>6Interestingly, non-chair ring puckering of carbohydrates has been found to play a role in various processes: this includes the reaction paths of glycoside hydrolases, where carbohydrate substrates exhibit conformational itineraries through a range of non-chair puckered forms. ADDIN REFMGR.CITE <Refman><Cite><Author>Speciale</Author><Year>2014</Year><RecNum>1123</RecNum><IDText>Dissecting conformational contributions to glycosidase catalysis and inhibition</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1123</Ref_ID><Title_Primary>Dissecting conformational contributions to glycosidase catalysis and inhibition</Title_Primary><Authors_Primary>Speciale,Gaetano</Authors_Primary><Authors_Primary>Thompson,Andrew J.</Authors_Primary><Authors_Primary>Davies,Gideon J.</Authors_Primary><Authors_Primary>Williams,Spencer J.</Authors_Primary><Date_Primary>2014</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1</Start_Page><End_Page>13</End_Page><Periodical>Current opinion in structural biology</Periodical><Volume>28</Volume><ZZ_JournalStdAbbrev><f name="System">Current opinion in structural biology</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>7 Similarly, adoption of the skew-boat 2SO ring conformer in L-iduronic residues of the anticoagulant GAG, heparin, is essential in its activation of antithrombin III. ADDIN REFMGR.CITE <Refman><Cite><Author>Das</Author><Year>2001</Year><RecNum>1124</RecNum><IDText>Synthesis of Conformationally Locked lG??Iduronic Acid Derivatives: Direct Evidence for a Critical Role of the SkewG??Boat 2S0 Conformer in the Activation of Antithrombin by Heparin</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1124</Ref_ID><Title_Primary>Synthesis of Conformationally Locked l<f name="Symbol">G</f>ÇÉIduronic Acid Derivatives: Direct Evidence for a Critical Role of the Skew<f name="Symbol">G</f>ÇÉBoat 2S0 Conformer in the Activation of Antithrombin by Heparin</Title_Primary><Authors_Primary>Das,Sanjoy K.</Authors_Primary><Authors_Primary>Mallet,Jean<f name="Symbol">G</f>ÇÉMaurice</Authors_Primary><Authors_Primary>Esnault,Jacques</Authors_Primary><Authors_Primary>Driguez,Pierre<f name="Symbol">G</f>ÇÉAlexandre</Authors_Primary><Authors_Primary>Duchaussoy,Philippe</Authors_Primary><Authors_Primary>Sizun,Philippe</Authors_Primary><Authors_Primary>Herault,Jean<f name="Symbol">G</f>ÇÉPascal</Authors_Primary><Authors_Primary>Herbert,Jean<f name="Symbol">G</f>ÇÉMarc</Authors_Primary><Authors_Primary>PETITOU,Maurice</Authors_Primary><Authors_Primary>Sina++,Pierre</Authors_Primary><Date_Primary>2001</Date_Primary><Keywords>ACID</Keywords><Keywords>ACTIVATION</Keywords><Reprint>Not in File</Reprint><Start_Page>4821</Start_Page><End_Page>4834</End_Page><Periodical>Chemistry<f name="Symbol">G</f>ÇôA European Journal</Periodical><Volume>7</Volume><Issue>22</Issue><ZZ_JournalStdAbbrev><f name="System">Chemistry</f><f name="Symbol">G</f><f name="System">ÇôA European Journal</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>8 As another example, the ?-GlcNAc residue of Lewis X is observed to assume a OS2 pucker on binding to the lectin protein, BambL.PFJlZm1hbj48Q2l0ZT48QXV0aG9yPlRvcGluPC9BdXRob3I+PFllYXI+MjAxMzwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 9 Therefore, knowledge of the relative stability of different ring pucker conformations of constituent monosaccharide residues can assist in understanding carbohydrate interactions and reactivity. However, to characterize the ring puckering free energy landscape of carbohydrates is non-trivial: it is challenging to study by NMR methods due to the microsecond timescale of pucker transitions and the low population of rare but important pucker states; ADDIN REFMGR.CITE <Refman><Cite><Author>Plazinski</Author><Year>2015</Year><RecNum>1127</RecNum><IDText>Kinetic characteristics of conformational changes in the hexopyranose rings</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1127</Ref_ID><Title_Primary>Kinetic characteristics of conformational changes in the hexopyranose rings</Title_Primary><Authors_Primary>Plazinski,Wojciech</Authors_Primary><Authors_Primary>Drach,Mateusz</Authors_Primary><Date_Primary>2015</Date_Primary><Keywords>conformational change</Keywords><Keywords>CONFORMATIONAL-CHANGES</Keywords><Keywords>RING</Keywords><Reprint>Not in File</Reprint><Start_Page>41</Start_Page><End_Page>50</End_Page><Periodical>Carbohydr.Res.</Periodical><Volume>416</Volume><ZZ_JournalStdAbbrev><f name="System">Carbohydr.Res.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Woods</Author><Year>2018</Year><RecNum>1166</RecNum><IDText>Predicting the Structures of Glycans, Glycoproteins, and Their Complexes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1166</Ref_ID><Title_Primary>Predicting the Structures of Glycans, Glycoproteins, and Their Complexes</Title_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Date_Primary>2018/9/12</Date_Primary><Keywords>COMPLEX</Keywords><Keywords>COMPLEXES</Keywords><Keywords>Structure</Keywords><Reprint>Not in File</Reprint><Start_Page>8005</Start_Page><End_Page>8024</End_Page><Periodical>Chem.Rev.</Periodical><Volume>118</Volume><Issue>17</Issue><Web_URL> name="System">Chemical Reviews</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Chem.Rev.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>10,11 it is also difficult to examine ring puckering computationally due to the high energy barriers separating stable conformers, necessitating multi-microsecond molecular dynamics (MD) simulations. ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2010</Year><RecNum>1113</RecNum><IDText>Free energy landscapes of iduronic acid and related monosaccharides</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1113</Ref_ID><Title_Primary>Free energy landscapes of iduronic acid and related monosaccharides</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Hansen,Steen U.</Authors_Primary><Authors_Primary>Gardiner,John</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>ACID</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Reprint>Not in File</Reprint><Start_Page>13132</Start_Page><End_Page>13134</End_Page><Periodical>J.Am.Chem.Soc.</Periodical><Volume>132</Volume><Issue>38</Issue><ZZ_JournalStdAbbrev><f name="System">J.Am.Chem.Soc.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>DeMarco</Author><Year>2008</Year><RecNum>1156</RecNum><IDText>Structural glycobiology: a game of snakes and ladders</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1156</Ref_ID><Title_Primary>Structural glycobiology: a game of snakes and ladders</Title_Primary><Authors_Primary>DeMarco,Mari L.</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Date_Primary>2008</Date_Primary><Reprint>Not in File</Reprint><Start_Page>426</Start_Page><End_Page>440</End_Page><Periodical>glycob</Periodical><Volume>18</Volume><Issue>6</Issue><ZZ_JournalFull><f name="System">GLYCOBIOLOGY</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">glycob</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>12,13 Consequently, enhanced sampling MD techniques have been applied to the challenge of exploring carbohydrate ring pucker; this includes the adaptive reaction coordinate force method, ADDIN REFMGR.CITE <Refman><Cite><Author>Naidoo</Author><Year>2011</Year><RecNum>1133</RecNum><IDText>FEARCF a multidimensional free energy method for investigating conformational landscapes and chemical reaction mechanisms</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1133</Ref_ID><Title_Primary>FEARCF a multidimensional free energy method for investigating conformational landscapes and chemical reaction mechanisms</Title_Primary><Authors_Primary>Naidoo,Kevin J.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>ASSOCIATION</Keywords><Keywords>COMPLEX</Keywords><Keywords>COMPLEXES</Keywords><Keywords>CONFORMATION</Keywords><Keywords>EFFICIENCY</Keywords><Keywords>EFFICIENT</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Keywords>IMPLEMENTATION</Keywords><Keywords>MEAN FORCE</Keywords><Keywords>MECHANISM</Keywords><Keywords>PREPHENATE</Keywords><Keywords>puckering</Keywords><Keywords>RING</Keywords><Keywords>SIMULATION</Keywords><Keywords>SIMULATIONS</Keywords><Keywords>SURFACE</Keywords><Keywords>VOLUMES</Keywords><Reprint>Not in File</Reprint><Start_Page>1962</Start_Page><End_Page>1973</End_Page><Periodical>Science China Chemistry</Periodical><Volume>54</Volume><Issue>12</Issue><Web_URL> name="System">Science China Chemistry</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>14 metadynamicsPFJlZm1hbj48Q2l0ZT48QXV0aG9yPkF1dGllcmk8L0F1dGhvcj48WWVhcj4yMDEwPC9ZZWFyPjxS
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ADDIN EN.CITE.DATA 19,20,21,22 To date, however, enhanced sampling MD methods have not been deployed to examine the ring puckering of GAG monosaccharides. Recently, we introduced the msesMD method (multi-dimensional swarm enhanced sampling molecular dynamics) as an intuitive and effective biased MD approach. ADDIN REFMGR.CITE <Refman><Cite><Author>Alibay</Author><Year>2018</Year><RecNum>1121</RecNum><IDText>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1121</Ref_ID><Title_Primary>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</Title_Primary><Authors_Primary>Alibay,Irfan</Authors_Primary><Authors_Primary>Burusco,Kepa K.</Authors_Primary><Authors_Primary>Bruce,Neil J.</Authors_Primary><Authors_Primary>Bryce,Richard A.</Authors_Primary><Date_Primary>2018/3/8</Date_Primary><Keywords>AQUEOUS-SOLUTION</Keywords><Keywords>DYNAMICS</Keywords><Keywords>IDENTIFICATION</Keywords><Keywords>molecular dynamics</Keywords><Keywords>MOLECULAR-DYNAMICS</Keywords><Reprint>Not in File</Reprint><Start_Page>2462</Start_Page><End_Page>2474</End_Page><Periodical>J.Phys.Chem.B</Periodical><Volume>122</Volume><Issue>9</Issue><Web_URL> name="System">J.Phys.Chem.B</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>23 The msesMD method involves the coupling of a swarm of simulation replicas via attractive and repulsive potentials acting on dihedral angles of interest.PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkFsaWJheTwvQXV0aG9yPjxZZWFyPjIwMTg8L1llYXI+PFJl
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ADDIN EN.CITE.DATA 23,24,25 We demonstrated the computational efficiency of msesMD simulations in sampling Lewis tri- and tetrasaccharide conformations separated by high energy barriers to rotation about glycosidic torsions. ADDIN REFMGR.CITE <Refman><Cite><Author>Alibay</Author><Year>2018</Year><RecNum>1121</RecNum><IDText>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1121</Ref_ID><Title_Primary>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</Title_Primary><Authors_Primary>Alibay,Irfan</Authors_Primary><Authors_Primary>Burusco,Kepa K.</Authors_Primary><Authors_Primary>Bruce,Neil J.</Authors_Primary><Authors_Primary>Bryce,Richard A.</Authors_Primary><Date_Primary>2018/3/8</Date_Primary><Keywords>AQUEOUS-SOLUTION</Keywords><Keywords>DYNAMICS</Keywords><Keywords>IDENTIFICATION</Keywords><Keywords>molecular dynamics</Keywords><Keywords>MOLECULAR-DYNAMICS</Keywords><Reprint>Not in File</Reprint><Start_Page>2462</Start_Page><End_Page>2474</End_Page><Periodical>J.Phys.Chem.B</Periodical><Volume>122</Volume><Issue>9</Issue><Web_URL> name="System">J.Phys.Chem.B</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>23 In this work, we apply msesMD simulations to characterize the free energy landscape of ring pucker in GAG-related monosaccharides. We first validate the msesMD approach against unbiased multi-microsecond MD simulations for four unmodified monosaccharides: the anomers, α-D-glucose (α-Glc) and β-D-glucose (?-Glc); and the uronic acid epimers, α-L-iduronic acid (IdoA) and β-D-glucuronic acid (GlcA). Having established the efficient and quantitative sampling of ring pucker populations for these monosaccharides by msesMD simulations, we then employ this method to study a range of N-acetylated and sulfated GAG residues of IdoA, GlcA, N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) (Figure 1). For simulating these monosaccharides, we employ the GLYCAM06 force field ADDIN REFMGR.CITE <Refman><Cite><Author>Kirschner</Author><Year>2008</Year><RecNum>1143</RecNum><IDText>GLYCAM06: a generalizable biomolecular force field. Carbohydrates</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1143</Ref_ID><Title_Primary>GLYCAM06: a generalizable biomolecular force field. Carbohydrates</Title_Primary><Authors_Primary>Kirschner,Karl N.</Authors_Primary><Authors_Primary>Yongye,Austin B.</Authors_Primary><Authors_Primary>Tschampel,Sarah M.</Authors_Primary><Authors_Primary>Gonz+ílez<f name="Symbol">G</f>ÇÉOuteiri+¦o,Jorge</Authors_Primary><Authors_Primary>Daniels,Charlisa R.</Authors_Primary><Authors_Primary>Foley,B.Lachele</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>FORCE FIELD</Keywords><Keywords>FORCE-FIELD</Keywords><Keywords>GLYCAM06</Keywords><Reprint>Not in File</Reprint><Start_Page>622</Start_Page><End_Page>655</End_Page><Periodical>put.Chem.</Periodical><Volume>29</Volume><Issue>4</Issue><ZZ_JournalStdAbbrev><f name="System">put.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>26 and its recently introduced extension for modelling GAGs. ADDIN REFMGR.CITE <Refman><Cite><Author>Singh</Author><Year>2016</Year><RecNum>1142</RecNum><IDText>Extension and validation of the GLYCAM force field parameters for modeling glycosaminoglycans</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1142</Ref_ID><Title_Primary>Extension and validation of the GLYCAM force field parameters for modeling glycosaminoglycans</Title_Primary><Authors_Primary>Singh,Arunima</Authors_Primary><Authors_Primary>Tessier,Matthew B.</Authors_Primary><Authors_Primary>Pederson,Kari</Authors_Primary><Authors_Primary>Wang,Xiaocong</Authors_Primary><Authors_Primary>Venot,Andre P.</Authors_Primary><Authors_Primary>Boons,Geert Jan</Authors_Primary><Authors_Primary>Prestegard,James H.</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Date_Primary>2016/2/9</Date_Primary><Keywords>FORCE FIELD</Keywords><Keywords>FORCE-FIELD</Keywords><Keywords>PARAMETERS</Keywords><Keywords>VALIDATION</Keywords><Reprint>Not in File</Reprint><Start_Page>927</Start_Page><End_Page>935</End_Page><Periodical>Can.J.Chem.</Periodical><Volume>94</Volume><Issue>11</Issue><Web_URL> name="System">Canadian Journal of Chemistry</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Can.J.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>27 Thus, we employ enhanced sampling molecular dynamics simulations to examine the puckering landscapes of sulfated monosaccharides, residues which feature in biologically key GAGs such as heparan sulfate/heparin, dermatan sulfate and chondroitin sulfate.PFJlZm1hbj48Q2l0ZT48QXV0aG9yPlNhdHRlbGxlPC9BdXRob3I+PFllYXI+MjAxMTwvWWVhcj48
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ADDIN EN.CITE.DATA 31 Carbohydrate parameters were obtained from the GLYCAM06 (version j-1) force field, ADDIN REFMGR.CITE <Refman><Cite><Author>Kirschner</Author><Year>2008</Year><RecNum>1143</RecNum><IDText>GLYCAM06: a generalizable biomolecular force field. Carbohydrates</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1143</Ref_ID><Title_Primary>GLYCAM06: a generalizable biomolecular force field. Carbohydrates</Title_Primary><Authors_Primary>Kirschner,Karl N.</Authors_Primary><Authors_Primary>Yongye,Austin B.</Authors_Primary><Authors_Primary>Tschampel,Sarah M.</Authors_Primary><Authors_Primary>Gonz+ílez<f name="Symbol">G</f>ÇÉOuteiri+¦o,Jorge</Authors_Primary><Authors_Primary>Daniels,Charlisa R.</Authors_Primary><Authors_Primary>Foley,B.Lachele</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>FORCE FIELD</Keywords><Keywords>FORCE-FIELD</Keywords><Keywords>GLYCAM06</Keywords><Reprint>Not in File</Reprint><Start_Page>622</Start_Page><End_Page>655</End_Page><Periodical>put.Chem.</Periodical><Volume>29</Volume><Issue>4</Issue><ZZ_JournalStdAbbrev><f name="System">put.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>26 with additional parameters for the unsubstituted and N-sulfated glucosamine units obtained from a separate GLYCAM06 release for glycosaminoglycan monosaccharides. ADDIN REFMGR.CITE <Refman><Cite><Author>Singh</Author><Year>2016</Year><RecNum>1142</RecNum><IDText>Extension and validation of the GLYCAM force field parameters for modeling glycosaminoglycans</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1142</Ref_ID><Title_Primary>Extension and validation of the GLYCAM force field parameters for modeling glycosaminoglycans</Title_Primary><Authors_Primary>Singh,Arunima</Authors_Primary><Authors_Primary>Tessier,Matthew B.</Authors_Primary><Authors_Primary>Pederson,Kari</Authors_Primary><Authors_Primary>Wang,Xiaocong</Authors_Primary><Authors_Primary>Venot,Andre P.</Authors_Primary><Authors_Primary>Boons,Geert Jan</Authors_Primary><Authors_Primary>Prestegard,James H.</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Date_Primary>2016/2/9</Date_Primary><Keywords>FORCE FIELD</Keywords><Keywords>FORCE-FIELD</Keywords><Keywords>PARAMETERS</Keywords><Keywords>VALIDATION</Keywords><Reprint>Not in File</Reprint><Start_Page>927</Start_Page><End_Page>935</End_Page><Periodical>Can.J.Chem.</Periodical><Volume>94</Volume><Issue>11</Issue><Web_URL> name="System">Canadian Journal of Chemistry</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Can.J.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>27 O-sulfated glycosaminoglycan monosaccharide parameters were set using the GLYCAM06 transferable sulfation method as described by Singh et al. ADDIN REFMGR.CITE <Refman><Cite><Author>Singh</Author><Year>2016</Year><RecNum>1142</RecNum><IDText>Extension and validation of the GLYCAM force field parameters for modeling glycosaminoglycans</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1142</Ref_ID><Title_Primary>Extension and validation of the GLYCAM force field parameters for modeling glycosaminoglycans</Title_Primary><Authors_Primary>Singh,Arunima</Authors_Primary><Authors_Primary>Tessier,Matthew B.</Authors_Primary><Authors_Primary>Pederson,Kari</Authors_Primary><Authors_Primary>Wang,Xiaocong</Authors_Primary><Authors_Primary>Venot,Andre P.</Authors_Primary><Authors_Primary>Boons,Geert Jan</Authors_Primary><Authors_Primary>Prestegard,James H.</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Date_Primary>2016/2/9</Date_Primary><Keywords>FORCE FIELD</Keywords><Keywords>FORCE-FIELD</Keywords><Keywords>PARAMETERS</Keywords><Keywords>VALIDATION</Keywords><Reprint>Not in File</Reprint><Start_Page>927</Start_Page><End_Page>935</End_Page><Periodical>Can.J.Chem.</Periodical><Volume>94</Volume><Issue>11</Issue><Web_URL> name="System">Canadian Journal of Chemistry</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Can.J.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>27 The various monosaccharides (Figure 1) were built in the 4C1 ring configuration, with hydroxyl substituents at the C1 position. The systems were neutralised using sodium ions where appropriate and explicitly solvated in truncated octahedron boxes with TIP3P ADDIN REFMGR.CITE <Refman><Cite><Author>Jorgensen</Author><Year>1983</Year><RecNum>14</RecNum><IDText>Comparison of simple potential functions for simulating liquid water</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>14</Ref_ID><Title_Primary>Comparison of simple potential functions for simulating liquid water</Title_Primary><Authors_Primary>Jorgensen,W.L.</Authors_Primary><Authors_Primary>Chandrasekhar,J.</Authors_Primary><Authors_Primary>Madura,J.D.</Authors_Primary><Authors_Primary>Impey,R.W.</Authors_Primary><Authors_Primary>Klein,M.L.</Authors_Primary><Date_Primary>1983</Date_Primary><Reprint>Not in File</Reprint><Start_Page>926</Start_Page><End_Page>935</End_Page><Periodical>J.Chem.Phys.</Periodical><Volume>79</Volume><ZZ_JournalStdAbbrev><f name="System">J.Chem.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>32 waters. For β-GalNAc and its sulfated decorations (i.e. β-GalNAc(4S), β-GalNAc(6S), and β-GalNAc(4S, 6S)), an extra set of simulations was carried out at an ionic concentration of approximately between 150 and 175 mM, with 3 to 4 pairs of sodium and chloride ions added to the simulation box.MD simulations. All simulations were equilibrated using the following protocol. Solvated monosaccharide boxes were minimised and then heated from 0 to 298 K over 500 ps under constant volume conditions (NVT). The box density was then equilibrated to a target pressure of 1 bar via 1 ns of constant pressure (NPT) simulation using the Monte Carlo barostat, ADDIN REFMGR.CITE <Refman><Cite><Author>Allen</Author><Year>1987</Year><RecNum>239</RecNum><IDText>Computer simulation of liquids</IDText><MDL Ref_Type="Book, Whole"><Ref_Type>Book, Whole</Ref_Type><Ref_ID>239</Ref_ID><Title_Primary>Computer simulation of liquids</Title_Primary><Authors_Primary>Allen,M.P.</Authors_Primary><Authors_Primary>Tildesley,D.J.</Authors_Primary><Date_Primary>1987</Date_Primary><Reprint>Not in File</Reprint><Publisher>OUP</Publisher><ZZ_WorkformID>2</ZZ_WorkformID></MDL></Cite></Refman>33 with volume exchange attempts every 100 steps. The system was then further equilibrated under NVT conditions for a further 1 ns. All subsequent simulations were carried out in the NVT ensemble. Temperature control was achieved using the Langevin thermostat, ADDIN REFMGR.CITE <Refman><Cite><Author>Allen</Author><Year>1987</Year><RecNum>239</RecNum><IDText>Computer simulation of liquids</IDText><MDL Ref_Type="Book, Whole"><Ref_Type>Book, Whole</Ref_Type><Ref_ID>239</Ref_ID><Title_Primary>Computer simulation of liquids</Title_Primary><Authors_Primary>Allen,M.P.</Authors_Primary><Authors_Primary>Tildesley,D.J.</Authors_Primary><Date_Primary>1987</Date_Primary><Reprint>Not in File</Reprint><Publisher>OUP</Publisher><ZZ_WorkformID>2</ZZ_WorkformID></MDL></Cite><Cite><Author>Lemons</Author><Year>1997</Year><RecNum>1160</RecNum><IDText>Paul Langevin's 1908 paper "On the theory of Brownian motion / Sur la theorie du mouvement brownien", [CR Acad. Sci.(Paris) 146, 530-533 (1908)]</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1160</Ref_ID><Title_Primary>Paul Langevin's 1908 paper "On the theory of Brownian motion<f name="Symbol"> / </f>Sur la theorie du mouvement brownien", <f name="Symbol">[</f>CR Acad. Sci.(Paris) 146, 530<f name="Symbol">-</f>533 (1908)]</Title_Primary><Authors_Primary>Lemons,Don S.</Authors_Primary><Authors_Primary>Gythiel,Anthony</Authors_Primary><Date_Primary>1997</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1079</Start_Page><End_Page>1081</End_Page><Periodical>Am.J.Phys.</Periodical><Volume>65</Volume><Issue>11</Issue><ZZ_JournalStdAbbrev><f name="System">Am.J.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>33,34 with a collision frequency of 3 ps-1 and a target temperature of 298 K. Hydrogen bond motion was constrained using the SHAKE ADDIN REFMGR.CITE <Refman><Cite><Author>Ryckaert</Author><Year>1977</Year><RecNum>4</RecNum><IDText>Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>4</Ref_ID><Title_Primary>Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes</Title_Primary><Authors_Primary>Ryckaert,J.P.</Authors_Primary><Authors_Primary>Ciccotti,G.</Authors_Primary><Authors_Primary>Berendsen,H.J.C.</Authors_Primary><Date_Primary>1977</Date_Primary><Reprint>Not in File</Reprint><Start_Page>327</Start_Page><End_Page>341</End_Page><Periodical>put.Phys.</Periodical><Volume>23</Volume><ZZ_JournalStdAbbrev><f name="System">put.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>35 and SETTLE ADDIN REFMGR.CITE <Refman><Cite><Author>Miyamoto</Author><Year>1992</Year><RecNum>1145</RecNum><IDText>Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1145</Ref_ID><Title_Primary>Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models</Title_Primary><Authors_Primary>Miyamoto,Shuichi</Authors_Primary><Authors_Primary>Kollman,Peter A.</Authors_Primary><Date_Primary>1992</Date_Primary><Keywords>ALGORITHM</Keywords><Keywords>MODEL</Keywords><Keywords>MODELS</Keywords><Keywords>water</Keywords><Keywords>WATER MODEL</Keywords><Reprint>Not in File</Reprint><Start_Page>952</Start_Page><End_Page>962</End_Page><Periodical>put.Chem.</Periodical><Volume>13</Volume><Issue>8</Issue><ZZ_JournalStdAbbrev><f name="System">put.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>36 algorithms for solute and water molecule respectively. A 2 fs integration timestep was used for all msesMD simulations, whilst the unbiased MD simulations employed the hydrogen mass repartitioning (HMR) ADDIN REFMGR.CITE <Refman><Cite><Author>Feenstra</Author><Year>1999</Year><RecNum>1146</RecNum><IDText>Improving efficiency of large time-scale molecular dynamics simulations of hydrogen-rich systems</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1146</Ref_ID><Title_Primary>Improving efficiency of large time<f name="Symbol">-</f>scale molecular dynamics simulations of hydrogen<f name="Symbol">-</f>rich systems</Title_Primary><Authors_Primary>Feenstra,K.Anton</Authors_Primary><Authors_Primary>Hess,Berk</Authors_Primary><Authors_Primary>Berendsen,Herman JC</Authors_Primary><Date_Primary>1999</Date_Primary><Keywords>DYNAMICS</Keywords><Keywords>DYNAMICS SIMULATION</Keywords><Keywords>DYNAMICS SIMULATIONS</Keywords><Keywords>EFFICIENCY</Keywords><Keywords>molecular dynamics</Keywords><Keywords>molecular dynamics simulation</Keywords><Keywords>molecular dynamics simulations</Keywords><Keywords>MOLECULAR-DYNAMICS</Keywords><Keywords>SIMULATION</Keywords><Keywords>SIMULATIONS</Keywords><Keywords>SYSTEMS</Keywords><Reprint>Not in File</Reprint><Start_Page>786</Start_Page><End_Page>798</End_Page><Periodical>put.Chem.</Periodical><Volume>20</Volume><Issue>8</Issue><ZZ_JournalStdAbbrev><f name="System">put.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>37 methodology with a timestep of 4 fs. Following Hopkins et al., ADDIN REFMGR.CITE <Refman><Cite><Author>Hopkins</Author><Year>2015</Year><RecNum>1147</RecNum><IDText>Long-time-step molecular dynamics through hydrogen mass repartitioning</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1147</Ref_ID><Title_Primary>Long-time-step molecular dynamics through hydrogen mass repartitioning</Title_Primary><Authors_Primary>Hopkins,Chad W.</Authors_Primary><Authors_Primary>Le Grand,Scott</Authors_Primary><Authors_Primary>Walker,Ross C.</Authors_Primary><Authors_Primary>Roitberg,Adrian E.</Authors_Primary><Date_Primary>2015</Date_Primary><Keywords>DYNAMICS</Keywords><Keywords>HYDROGEN</Keywords><Keywords>molecular dynamics</Keywords><Keywords>MOLECULAR-DYNAMICS</Keywords><Reprint>Not in File</Reprint><Start_Page>1864</Start_Page><End_Page>1874</End_Page><Periodical>J.Chem.Theory Comput.</Periodical><Volume>11</Volume><Issue>4</Issue><ZZ_JournalStdAbbrev><f name="System">J.Chem.Theory Comput.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>38 2 amu was repartitioned from heavy atoms to hydrogens via the AMBER parmed utility. A 9 ? cut-off was used for short range non-bonded interactions, with long range electrostatics calculated via the particle mesh Ewald approach. ADDIN REFMGR.CITE <Refman><Cite><Author>Essmann</Author><Year>1995</Year><RecNum>16</RecNum><IDText>A smooth particle mesh Ewald method</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>16</Ref_ID><Title_Primary>A smooth particle mesh Ewald method</Title_Primary><Authors_Primary>Essmann,U.</Authors_Primary><Authors_Primary>Perera,L.</Authors_Primary><Authors_Primary>Berkowitz,M.L.</Authors_Primary><Authors_Primary>Darden,T.</Authors_Primary><Authors_Primary>Lee,H.</Authors_Primary><Authors_Primary>Pedersen,L.G.</Authors_Primary><Date_Primary>1995</Date_Primary><Reprint>Not in File</Reprint><Start_Page>8577</Start_Page><End_Page>8593</End_Page><Periodical>J.Chem.Phys.</Periodical><Volume>103</Volume><ZZ_JournalStdAbbrev><f name="System">J.Chem.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Darden</Author><Year>1993</Year><RecNum>490</RecNum><IDText>Particle mesh Ewald: An N.log(N) method for Ewald sums in large systems.</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>490</Ref_ID><Title_Primary>Particle mesh Ewald: An N.log(N) method for Ewald sums in large systems.</Title_Primary><Authors_Primary>Darden,T.</Authors_Primary><Authors_Primary>York,D.M.</Authors_Primary><Authors_Primary>Pedersen,L.</Authors_Primary><Date_Primary>1993</Date_Primary><Reprint>Not in File</Reprint><Start_Page>10089</Start_Page><End_Page>10092</End_Page><Periodical>J.Chem.Phys.</Periodical><Volume>98</Volume><ZZ_JournalStdAbbrev><f name="System">J.Chem.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>39,40 Starting from the equilibrated systems, individual production simulations were propagated for a total of 10 ?s, with configurations sampled every 5 ps. For α-Glc and GlcA, simulations were extended by a further 10 ?s due to poor sampling of chair interconversion events.msesMD simulations. The same overall msesMD simulation approach employed in our recent work was used here. ADDIN REFMGR.CITE <Refman><Cite><Author>Alibay</Author><Year>2018</Year><RecNum>1121</RecNum><IDText>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1121</Ref_ID><Title_Primary>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</Title_Primary><Authors_Primary>Alibay,Irfan</Authors_Primary><Authors_Primary>Burusco,Kepa K.</Authors_Primary><Authors_Primary>Bruce,Neil J.</Authors_Primary><Authors_Primary>Bryce,Richard A.</Authors_Primary><Date_Primary>2018/3/8</Date_Primary><Keywords>AQUEOUS-SOLUTION</Keywords><Keywords>DYNAMICS</Keywords><Keywords>IDENTIFICATION</Keywords><Keywords>molecular dynamics</Keywords><Keywords>MOLECULAR-DYNAMICS</Keywords><Reprint>Not in File</Reprint><Start_Page>2462</Start_Page><End_Page>2474</End_Page><Periodical>J.Phys.Chem.B</Periodical><Volume>122</Volume><Issue>9</Issue><Web_URL> name="System">J.Phys.Chem.B</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>23 Briefly, eight replicas were coupled using our previously published swarm biasing potential with the parameters A = 3.195 kcal/mol, B = 2.625 rad-1, C = 0.75 kcal/mol and D = 0.5 rad-1. For all monosaccharides, the msesMD potential was applied to two non-overlapping ring torsions, O5-C1-C2-C3 and C3-C4-C5-O5. These boost coordinates were chosen due to their successful previous use in Hamiltonian replica exchange studies of uronic acid puckering by Babin and Sagui. ADDIN REFMGR.CITE <Refman><Cite><Author>Babin</Author><Year>2010</Year><RecNum>1131</RecNum><IDText>Conformational free energies of methyl-a-L-iduronic and methyl-b-D-glucuronic acids in water</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1131</Ref_ID><Title_Primary>Conformational free energies of methyl-<f name="Symbol">a</f>-L-iduronic and methyl-<f name="Symbol">b</f>-D-glucuronic acids in water</Title_Primary><Authors_Primary>Babin,Volodymyr</Authors_Primary><Authors_Primary>Sagui,Celeste</Authors_Primary><Date_Primary>2010/3/11</Date_Primary><Keywords>ACID</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Keywords>water</Keywords><Reprint>Not in File</Reprint><Start_Page>104108</Start_Page><Periodical>J.Chem.Phys.</Periodical><Volume>132</Volume><Issue>10</Issue><Web_URL> name="System">J.Chem.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>19 It is however noted that other puckering coordinates, such as Hill-Reilly ADDIN REFMGR.CITE <Refman><Cite><Author>Hill</Author><Year>2007</Year><RecNum>1148</RecNum><IDText>Puckering coordinates of monocyclic rings by triangular decomposition</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1148</Ref_ID><Title_Primary>Puckering coordinates of monocyclic rings by triangular decomposition</Title_Primary><Authors_Primary>Hill,Anthony D.</Authors_Primary><Authors_Primary>Reilly,Peter J.</Authors_Primary><Date_Primary>2007</Date_Primary><Keywords>decomposition</Keywords><Keywords>puckering</Keywords><Keywords>RING</Keywords><Reprint>Not in File</Reprint><Start_Page>1031</Start_Page><End_Page>1035</End_Page><Periodical>J.Chem.Inf.Model.</Periodical><Volume>47</Volume><Issue>3</Issue><ZZ_JournalFull><f name="System">Journal of Chemical Information and Modeling</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Chem.Inf.Model.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>41 or Cremer-Pople angles, ADDIN REFMGR.CITE <Refman><Cite><Author>Cremer</Author><Year>1975</Year><RecNum>1134</RecNum><IDText>General definition of ring puckering coordinates</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1134</Ref_ID><Title_Primary>General definition of ring puckering coordinates</Title_Primary><Authors_Primary>Cremer,D.</Authors_Primary><Authors_Primary>Pople,J.A.</Authors_Primary><Date_Primary>1975/3/1</Date_Primary><Keywords>puckering</Keywords><Keywords>RING</Keywords><Reprint>Not in File</Reprint><Start_Page>1354</Start_Page><End_Page>1358</End_Page><Periodical>J.Am.Chem.Soc.</Periodical><Volume>97</Volume><Issue>6</Issue><Web_URL> name="System">J.Am.Chem.Soc.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>6 have also been found to be effective in previous enhanced sampling studiesPFJlZm1hbj48Q2l0ZT48QXV0aG9yPk5haWRvbzwvQXV0aG9yPjxZZWFyPjIwMTE8L1llYXI+PFJl
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ADDIN EN.CITE.DATA 14,15 and could equally have been employed in the current work. On completing the unbiased MD equilibration process, eight independent trajectories were propagated over a 1 ns period. The msesMD potential was then slowly introduced across the replicas over a 600 ps period and the system allowed to re-equilibrate for a further 5.4 ns under the full influence of the potential. This was then followed by 195 ns per replica of production simulation with configurations sampled every picosecond.Analysis. Cremer-Pople θ and ? angles were calculated to characterise monosaccharide pucker. To quantify differences in occupation of ring conformations (1C4, 4C1, half-chair/envelope and boat/skew-boat), free energy surfaces were computed both as a function of θ and of θ and ?. For this, the relative Helmholtz free energy ΔΑ was computed from the normalised microstate probability density ρx according to ΔΑ = kBTlnρx, where kB is the Boltzmann constant and T is temperature. Estimates for ρx were obtained via the “counting approach” for unbiased MD simulations, ADDIN REFMGR.CITE <Refman><Cite><Author>Meirovitch</Author><Year>2007</Year><RecNum>1152</RecNum><IDText>Recent developments in methodologies for calculating the entropy and free energy of biological systems by computer simulation</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1152</Ref_ID><Title_Primary>Recent developments in methodologies for calculating the entropy and free energy of biological systems by computer simulation</Title_Primary><Authors_Primary>Meirovitch,Hagai</Authors_Primary><Date_Primary>2007</Date_Primary><Keywords>computer simulation</Keywords><Keywords>COMPUTER-SIMULATION</Keywords><Keywords>DYNAMICS</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Keywords>methodology</Keywords><Keywords>molecular dynamics</Keywords><Keywords>molecular dynamics method</Keywords><Keywords>MOLECULAR-DYNAMICS</Keywords><Keywords>PEPTIDE</Keywords><Keywords>POTENTIAL-ENERGY</Keywords><Keywords>PROTEIN</Keywords><Keywords>protein folding</Keywords><Keywords>PROTEINS</Keywords><Keywords>SIMULATION</Keywords><Keywords>SURFACE</Keywords><Keywords>SYSTEMS</Keywords><Keywords>Thermodynamic integration</Keywords><Reprint>Not in File</Reprint><Start_Page>181</Start_Page><End_Page>186</End_Page><Periodical>Curr.Opin.Struct.Biol.</Periodical><Volume>17</Volume><Issue>2</Issue><Web_URL> name="System">Curr.Opin.Struct.Biol.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>42 and for msesMD through the approach of Torrie and Valleau ADDIN REFMGR.CITE <Refman><Cite><Author>Torrie</Author><Year>1977</Year><RecNum>100</RecNum><IDText>Nonphysical sampling distributions in Monte Carlo free-energy estimation: Umbrella sampling</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>100</Ref_ID><Title_Primary>Nonphysical sampling distributions in Monte Carlo free-energy estimation: Umbrella sampling</Title_Primary><Authors_Primary>Torrie,G.M.</Authors_Primary><Authors_Primary>Valleau,J.P.</Authors_Primary><Date_Primary>1977</Date_Primary><Keywords>Monte Carlo</Keywords><Keywords>MONTE-CARLO</Keywords><Keywords>FREE-ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE ENERGY</Keywords><Reprint>Not in File</Reprint><Start_Page>187</Start_Page><End_Page>199</End_Page><Periodical>put.Phys.</Periodical><Volume>23</Volume><ZZ_JournalStdAbbrev><f name="System">put.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>43 as detailed previously.PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkFsaWJheTwvQXV0aG9yPjxZZWFyPjIwMTg8L1llYXI+PFJl
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ADDIN EN.CITE.DATA 23,25 A histogram bin size of 6 was used, with maximum energy cutoffs of 12 and 8 kcal/mol for the 1D and 2D surfaces respectively. Relative free energies of different puckering states (Tables 1 and 2) present the lowest free energy bin value in the 2D Cremer-Pople θ??range defining that particular puckering state. Ensemble average estimate errors for the msesMD simulations were calculated via bootstrap analysis, randomly resampling the simulation data from all frames 100,000 times and calculating the error as the standard deviation in the bin energy estimates across all resamples. ADDIN REFMGR.CITE <Refman><Cite><Author>Alibay</Author><Year>2018</Year><RecNum>1121</RecNum><IDText>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1121</Ref_ID><Title_Primary>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</Title_Primary><Authors_Primary>Alibay,Irfan</Authors_Primary><Authors_Primary>Burusco,Kepa K.</Authors_Primary><Authors_Primary>Bruce,Neil J.</Authors_Primary><Authors_Primary>Bryce,Richard A.</Authors_Primary><Date_Primary>2018/3/8</Date_Primary><Keywords>AQUEOUS-SOLUTION</Keywords><Keywords>DYNAMICS</Keywords><Keywords>IDENTIFICATION</Keywords><Keywords>molecular dynamics</Keywords><Keywords>MOLECULAR-DYNAMICS</Keywords><Reprint>Not in File</Reprint><Start_Page>2462</Start_Page><End_Page>2474</End_Page><Periodical>J.Phys.Chem.B</Periodical><Volume>122</Volume><Issue>9</Issue><Web_URL> name="System">J.Phys.Chem.B</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>23 From unbiased MD trajectories, stochastic transition matrices were constructed to examine the probability of moving from one pucker state to another, at a resolution of 5 ps. These analyses were performed via in-house python scripts, using the NumPy () and SciPy libraries (); and the cpptraj program from AmberTools16. ADDIN REFMGR.CITE <Refman><Cite><Author>Roe</Author><Year>2013</Year><RecNum>1150</RecNum><IDText>PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1150</Ref_ID><Title_Primary>PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data</Title_Primary><Authors_Primary>Roe,Daniel R.</Authors_Primary><Authors_Primary>Cheatham,Thomas E.</Authors_Primary><Date_Primary>2013/7/9</Date_Primary><Keywords>analysis</Keywords><Keywords>DYNAMICS</Keywords><Keywords>molecular dynamics</Keywords><Keywords>MOLECULAR-DYNAMICS</Keywords><Reprint>Not in File</Reprint><Start_Page>3084</Start_Page><End_Page>3095</End_Page><Periodical>J.Chem.Theory Comput.</Periodical><Volume>9</Volume><Issue>7</Issue><Web_URL> name="System">J.Chem.Theory Comput.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>44 3. Results and discussion3.1 Comparison of puckering free energy landscapes via MD and msesMDFirstly, we compute the puckering free energy profiles for four biological relevant and computationally well-studied monosaccharides, α-Glc, ?-Glc, α-L-IdoA and ?-D-GlcA (Figure 1). PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkJhYmluPC9BdXRob3I+PFllYXI+MjAxMDwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 10,18,19,45 These pucker profiles (Figure 2) were computed as a function of Cremer-Pople angle θ, a coordinate which describes the change in conformation from the 4C1 chair (θ 0°) through boat/skew-boat (θ 90°) to the 1C4 chair pucker (θ 180°). Unbiased MD studies of ring puckering in monosaccharides have previously suggested that simulation lengths of 5 - 10 μs are necessary to achieve converged puckering free energy profiles. ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2010</Year><RecNum>1113</RecNum><IDText>Free energy landscapes of iduronic acid and related monosaccharides</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1113</Ref_ID><Title_Primary>Free energy landscapes of iduronic acid and related monosaccharides</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Hansen,Steen U.</Authors_Primary><Authors_Primary>Gardiner,John</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>ACID</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Reprint>Not in File</Reprint><Start_Page>13132</Start_Page><End_Page>13134</End_Page><Periodical>J.Am.Chem.Soc.</Periodical><Volume>132</Volume><Issue>38</Issue><ZZ_JournalStdAbbrev><f name="System">J.Am.Chem.Soc.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Sattelle</Author><Year>2011</Year><RecNum>1118</RecNum><IDText>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1118</Ref_ID><Title_Primary>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2011</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1651</Start_Page><End_Page>1662</End_Page><Periodical>glycob</Periodical><Volume>21</Volume><Issue>12</Issue><ZZ_JournalFull><f name="System">GLYCOBIOLOGY</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">glycob</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>12,28 Interestingly, here we observe that explicit solvent MD simulations in excess of 15 ?s were required to obtain a converged computed free energy profile of α-Glc (Figure 2a). Examination of the sampling of the ? pucker angle for α-Glc (Figure 3a) indicates infrequent transitions between the preferred 4C1 chair and the higher energy 1C4 (? ~170°) states, via the boat/skew-boat pucker region at θ values of ~90°. The computed profiles for β-Glc and IdoA are broadly similar to that of α-Glc (Figure 2b,c); however, more frequent transitions to the 1C4 pucker are exhibited for these monosaccharides (Figure 3c,e). For GlcA, the computed barrier to the 1C4 pucker seems highest of the four monosaccharides (Figure 2d), with only two transient excursions to this conformation over the 20 ?s trajectory (Figure 3g). Despite the infrequent sampling of the 1C4 state, there is reasonable agreement in the glucose 4C1/1C4 energy preference computed here with previous enhanced sampling MD studies. From our 20 ?s unbiased MD simulations, the computed pucker landscapes indicate that for both ?-Glc and ?-Glc, the expected 4C1 chair conformer is favored (Figure 2a,b); the 4C1 preference is computed as 3.5 kcal/mol for ?-Glc, but only 0.2 kcal/mol for the ?-anomer (Table 1). This 3.3 kcal/mol reduction in 4C1 preference by ?-Glc is reflected by a MD study ADDIN REFMGR.CITE <Refman><Cite><Author>Wang</Author><Year>2018</Year><RecNum>1130</RecNum><IDText>Efficient sampling of puckering states of monosaccharides through replica exchange with solute tempering and bond softening</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1130</Ref_ID><Title_Primary>Efficient sampling of puckering states of monosaccharides through replica exchange with solute tempering and bond softening</Title_Primary><Authors_Primary>Wang,Lingle</Authors_Primary><Authors_Primary>Berne,B.J.</Authors_Primary><Date_Primary>2018/5/8</Date_Primary><Keywords>EFFICIENT</Keywords><Keywords>puckering</Keywords><Keywords>Replica Exchange</Keywords><Keywords>STATE</Keywords><Reprint>Not in File</Reprint><Start_Page>072306</Start_Page><Periodical>J.Chem.Phys.</Periodical><Volume>149</Volume><Issue>7</Issue><Web_URL> name="System">J.Chem.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>20 using replica exchange with solute tempering/bond softening (REST/BOS) in conjunction with the OPLS3 ADDIN REFMGR.CITE <Refman><Cite><Author>Harder</Author><Year>2016</Year><RecNum>1164</RecNum><IDText>OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1164</Ref_ID><Title_Primary>OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins</Title_Primary><Authors_Primary>Harder,Edward</Authors_Primary><Authors_Primary>Damm,Wolfgang</Authors_Primary><Authors_Primary>Maple,Jon</Authors_Primary><Authors_Primary>Wu,Chuanjie</Authors_Primary><Authors_Primary>Reboul,Mark</Authors_Primary><Authors_Primary>Xiang,Jin Yu</Authors_Primary><Authors_Primary>Wang,Lingle</Authors_Primary><Authors_Primary>Lupyan,Dmitry</Authors_Primary><Authors_Primary>Dahlgren,Markus K.</Authors_Primary><Authors_Primary>Knight,Jennifer L.</Authors_Primary><Authors_Primary>Kaus,Joseph W.</Authors_Primary><Authors_Primary>Cerutti,David S.</Authors_Primary><Authors_Primary>Krilov,Goran</Authors_Primary><Authors_Primary>Jorgensen,William L.</Authors_Primary><Authors_Primary>Abel,Robert</Authors_Primary><Authors_Primary>Friesner,Richard A.</Authors_Primary><Date_Primary>2016/1/12</Date_Primary><Keywords>FORCE FIELD</Keywords><Keywords>FORCE-FIELD</Keywords><Keywords>PROTEIN</Keywords><Keywords>PROTEINS</Keywords><Keywords>SMALL MOLECULES</Keywords><Reprint>Not in File</Reprint><Start_Page>281</Start_Page><End_Page>296</End_Page><Periodical>J.Chem.Theory Comput.</Periodical><Volume>12</Volume><Issue>1</Issue><Web_URL> name="System">J.Chem.Theory Comput.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>46 and SPC force fields; this study also found a reduction in stabilisation of 4C1 for ??Glc, by 2.2 kcal/mol, to the extent of 1C4 being the preferred state for the ?-anomer by 0.6 kcal/mol. Other in silico estimates yield a lower stability of the 1C4 state. In their MM3(92) modelling study, Dowd et al. obtained an energy difference of 5.2 kcal/mol. More recently, a free energy preference of 4.3 kcal/mol for 4C1 over 1C4 was computed for ?-Glc using the GROMOS 56A6CARBO force field. ADDIN REFMGR.CITE <Refman><Cite><Author>Plazinski</Author><Year>2016</Year><RecNum>1161</RecNum><IDText>Revision of the GROMOS 56A6CARBO force field: Improving the description of ring-conformational equilibria in hexopyranose-based carbohydrates chains</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1161</Ref_ID><Title_Primary>Revision of the GROMOS 56A6CARBO force field: Improving the description of ring-conformational equilibria in hexopyranose-based carbohydrates chains</Title_Primary><Authors_Primary>Plazinski,Wojciech</Authors_Primary><Authors_Primary>Lonardi,Alice</Authors_Primary><Authors_Primary>H++nenberger,Philippe H.</Authors_Primary><Date_Primary>2016/1/30</Date_Primary><Keywords>carbohydrate</Keywords><Keywords>CONFORMATION</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FLEXIBILITY</Keywords><Keywords>FORCE FIELD</Keywords><Keywords>FORCE-FIELD</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Keywords>GROMOS</Keywords><Keywords>GROMOS force field</Keywords><Keywords>hexopyranose</Keywords><Keywords>molecular dynamics</Keywords><Keywords>Property</Keywords><Keywords>RING</Keywords><Keywords>ring conformers</Keywords><Reprint>Not in File</Reprint><Start_Page>354</Start_Page><End_Page>365</End_Page><Periodical>put.Chem.</Periodical><Volume>37</Volume><Issue>3</Issue><Web_URL> name="System">put.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>47 Indeed, Angyal inferred from NMR experiments a difference in free energy of 4.2 kcal/mol favouring the 4C1 conformer over 1C4, ADDIN REFMGR.CITE <Refman><Cite><Author>Angyal</Author><Year>1968</Year><RecNum>1186</RecNum><IDText>Conformational analysis in carbohydrate chemistry. I. Conformational free energies. The conformations and &#945; : &#946; ratios of aldopyranoses in aqueous solution</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1186</Ref_ID><Title_Primary>Conformational analysis in carbohydrate chemistry. I. Conformational free energies. The conformations and &#945; : &#946; ratios of aldopyranoses in aqueous solution</Title_Primary><Authors_Primary>Angyal,S.J.</Authors_Primary><Date_Primary>1968</Date_Primary><Keywords>analysis</Keywords><Keywords>AQUEOUS-SOLUTION</Keywords><Keywords>carbohydrate</Keywords><Keywords>CONFORMATION</Keywords><Keywords>conformational analysis</Keywords><Keywords>CONFORMATIONAL-ANALYSIS</Keywords><Keywords>CONFORMATIONS</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Keywords>INTERACTION ENERGIES</Keywords><Reprint>Not in File</Reprint><Start_Page>2737</Start_Page><End_Page>2746</End_Page><Periodical>Australian Journal of Chemistry</Periodical><Volume>21</Volume><Issue>11</Issue><Web_URL> name="System">Australian Journal of Chemistry</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>48 albeit 1.8 kcal/mol less than for ?-Glc. For α-L-IdoA, our unbiased MD simulations predict its 4C1 and 1C4 forms are also similar in energy, with a preference for 1C4 of 0.3 kcal/mol (Figure 2c, Table 1). This is in accord with the observation of lability in IdoA ring pucker observed from previous MD simulation and NMR of the methyl aglycone. ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2010</Year><RecNum>1113</RecNum><IDText>Free energy landscapes of iduronic acid and related monosaccharides</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1113</Ref_ID><Title_Primary>Free energy landscapes of iduronic acid and related monosaccharides</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Hansen,Steen U.</Authors_Primary><Authors_Primary>Gardiner,John</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>ACID</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Reprint>Not in File</Reprint><Start_Page>13132</Start_Page><End_Page>13134</End_Page><Periodical>J.Am.Chem.Soc.</Periodical><Volume>132</Volume><Issue>38</Issue><ZZ_JournalStdAbbrev><f name="System">J.Am.Chem.Soc.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>12 For GlcA, the C5 epimer of IdoA, MD simulation predicts 4C1 chair as the dominant ring pucker conformation (Figure 2d), by 2.8 kcal/mol over the inverted chair structure (Table 1). This 4C1 preference is in accord with observation of GlcA residues in oligosaccharides from previous MD and NMR studies. ADDIN REFMGR.CITE <Refman><Cite><Author>Wang</Author><Year>2017</Year><RecNum>1116</RecNum><IDText>Synthesis of 3-O-sulfated oligosaccharides to understand the relationship between structures and functions of heparan sulfate</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1116</Ref_ID><Title_Primary>Synthesis of 3-O-sulfated oligosaccharides to understand the relationship between structures and functions of heparan sulfate</Title_Primary><Authors_Primary>Wang,Zhangjie</Authors_Primary><Authors_Primary>Hsieh,Po Hung</Authors_Primary><Authors_Primary>Xu,Yongmei</Authors_Primary><Authors_Primary>Thieker,David</Authors_Primary><Authors_Primary>Chai,Evangeline Juan En</Authors_Primary><Authors_Primary>Xie,Shaoshuai</Authors_Primary><Authors_Primary>Cooley,Brian</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Authors_Primary>Chi,Lianli</Authors_Primary><Authors_Primary>Liu,Jian</Authors_Primary><Date_Primary>2017</Date_Primary><Keywords>Structure</Keywords><Reprint>Not in File</Reprint><Start_Page>5249</Start_Page><End_Page>5256</End_Page><Periodical>Journal of the American Chemical Society</Periodical><Volume>139</Volume><Issue>14</Issue><ZZ_JournalFull><f name="System">Journal of the American Chemical Society</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>49, ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2010</Year><RecNum>1113</RecNum><IDText>Free energy landscapes of iduronic acid and related monosaccharides</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1113</Ref_ID><Title_Primary>Free energy landscapes of iduronic acid and related monosaccharides</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Hansen,Steen U.</Authors_Primary><Authors_Primary>Gardiner,John</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>ACID</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Reprint>Not in File</Reprint><Start_Page>13132</Start_Page><End_Page>13134</End_Page><Periodical>J.Am.Chem.Soc.</Periodical><Volume>132</Volume><Issue>38</Issue><ZZ_JournalStdAbbrev><f name="System">J.Am.Chem.Soc.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>12We next turn to consider ring pucker profiles for the four monosaccharides computed using msesMD enhanced sampling simulations. Free energy landscapes were determined based on msesMD simulation lengths of 45, 95, 145 and 195 ns (Figure 4); good convergence was found by 145 - 195 ns (Figure 4). Bootstrap sampling analysis of these 1D free energy profiles from 195 ns msesMD simulations (Supporting Information, Figure S1) indicates errors lying within 0.2 kcal/mol, although on occasion ranging up to 0.5 kcal/mol for very high energy half-chair and envelope transitional conformers. Overall, however, the free energy profiles recovered from the msesMD simulations appear well converged.Based on the 195 ns msesMD simulations, the free energy landscapes for the pucker conformations agree closely with the most exhaustive of the multi-microsecond unbiased MD simulations (Figure 2). For example, the profile of ?-Glc captures the relatively small 0.2 kcal/mol preference for the 4C1 form (Figure 2a, Table 1) that was predicted by the 15 and 20 ?s MD simulations but not 1, 5 and 10 ?s MD trajectories. The msesMD simulation of IdoA also correctly depicts a puckering free energy surface with a slight favoring of the 1C4 conformer (Figure 2c, Table 1). For the four sugars, agreement in the 4C1/1C4 energy difference between unbiased MD and msesMD simulations differs by at most 0.4 kcal/mol; this highest deviation is found for GlcA (Table 1). Indeed, the GlcA profile contains a high energy barrier of ~10 kcal/mol between boat/skew-boat forms and 1C4 (Figure 2d). Modelling the GlcA pucker profile appears to pose a challenge for the unbiased simulations; as described above, the 1C4 chair conformation region is only sampled twice for a few nanoseconds over the duration of the 20 ?s MD simulation (Figure 3g). This contrasts with comprehensive sampling by msesMD replicas over the 195 ns simulation (Figure 3h). To examine in more detail non-chair puckering states, we resolve the puckering free energy surface according to both Cremer-Pople angles, ? and ?. As for the free energy profiles based solely on??, the ???puckering profiles from msesMD have low bootstrap errors (Figure S2) and are in good agreement with unbiased MD predictions (Figure 5). The differing boat/skew-boat populations of the four monosaccharides on the puckering hypersurface is evident and reproduced by msesMD (Figure 5). The predicted identity of the lowest lying non-chair conformer agrees well in all four cases (Table 1). Interestingly, the epimers IdoA and GlcA exhibit a marked difference in preferred intermediate conformer, switching from 2SO for IdoA (Figure 5e,f) to 1S3 for GlcA (Figure 5g,h; Table 1). The ability of IdoA to readily access the 2SO conformer has been observed in previous MD simulations and NMR of oligosaccharides. ADDIN REFMGR.CITE <Refman><Cite><Author>Hsieh</Author><Year>2016</Year><RecNum>1115</RecNum><IDText>Uncovering the relationship between sulphation patterns and conformation of iduronic acid in heparan sulphate</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1115</Ref_ID><Title_Primary>Uncovering the relationship between sulphation patterns and conformation of iduronic acid in heparan sulphate</Title_Primary><Authors_Primary>Hsieh,Po Hung</Authors_Primary><Authors_Primary>Thieker,David F.</Authors_Primary><Authors_Primary>Guerrini,Marco</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Authors_Primary>Liu,Jian</Authors_Primary><Date_Primary>2016</Date_Primary><Keywords>ACID</Keywords><Keywords>CONFORMATION</Keywords><Reprint>Not in File</Reprint><Start_Page>29602</Start_Page><Periodical>Sci.Rep.</Periodical><Volume>6</Volume><ZZ_JournalStdAbbrev><f name="System">Sci.Rep.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Hricovini</Author><Year>2001</Year><RecNum>1120</RecNum><IDText>Conformation of heparin pentasaccharide bound to antithrombin III</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1120</Ref_ID><Title_Primary>Conformation of heparin pentasaccharide bound to antithrombin III</Title_Primary><Authors_Primary>Hricovini,M.</Authors_Primary><Authors_Primary>Guerrini,Marco</Authors_Primary><Authors_Primary>Bisio,Antonella</Authors_Primary><Authors_Primary>Torri,Giangiacomo</Authors_Primary><Authors_Primary>Petitou</Authors_Primary><Authors_Primary>Benito,C.A.S.U.</Authors_Primary><Date_Primary>2001</Date_Primary><Keywords>CONFORMATION</Keywords><Reprint>Not in File</Reprint><Start_Page>265</Start_Page><End_Page>272</End_Page><Periodical>Biochem.J.</Periodical><Volume>359</Volume><Issue>2</Issue><ZZ_JournalStdAbbrev><f name="System">Biochem.J.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>29,50 From our unbiased MD simulation of IdoA, we also observe that interconversion between conformers along the equator of its ???puckering hypersurface is occurring. To quantify this behavior, we construct a stochastic transition matrix from the 10 ?s trajectory of IdoA; we obtain probability values for the pairwise transitions from 2SO → 2,5B → 5S1 → B1,4 → 3S1 of 0.13, 0.04, 0.21 and 0.58 respectively (Supplemental Data). Interconversion between boat/skew-boat conformers is also observed for ?-Glc, ?-Glc and GlcA (Supplemental Data).For each of the four monosaccharides, the relative free energy of the lowest lying non-chair conformer from MD and msesMD agrees to within 0.1 kcal/mol (Table 1). The exception is for α-Glc, where a difference of 0.5 kcal/mol is found between MD and msesMD estimates for 1S5 (Table 1), a conformer which lies in the θ? region of (90°, 250-300°). In overall terms, however, there is good agreement between multi-microsecond MD and msesMD methods in predicted ? and ?? free energy profiles for ring puckering of α-Glc, ?-Glc, IdoA and GlcA. The detailed shifts in populated puckering states as a function of anomer and epimer are reproduced.3.2 Effect of sulfation on puckering free energy landscape Based on this assessment, we next apply our msesMD simulations to evaluate the pucker profiles of a range of biologically relevant monosaccharides derived from IdoA, GlcA, ??GlcNAc???-GlcNAc and ?-GalNAc residues with varying degrees of sulfation (Figure 1a). As before, an assessment of convergence in the free energy profiles computed by the msesMD simulations was performed; for the fifteen monosaccharide systems, the pucker free energy profiles typically had converged to within 0.2 kcal/mol by 195 ns (Figures S3-S7), with bootstrap errors also generally on the order of 0.2 kcal/mol (Figures S8-S17). Notable exceptions to this are β-Gal and β-Gal(6S), which show differences of up to 0.8 kcal/mol between the 145 and 195 ns profiles in the 1C4 region.We first consider the 2-O-sulfated forms of IdoA and GlcA, residues commonly found in GAGs; these are denoted IdoA2S and GlcA2S respectively (Figure 1a). The ring pucker profiles computed from 195 ns msesMD simulations predict that 2-O-sulfation of IdoA produces a subtle switch in preference (Figure 6a); this change is from a free energy difference of 0.5 kcal/mol favoring the 1C4 form over 4C1 in IdoA, to a 0.2 kcal/mol preference for the 4C1 over 1C4 pucker in IdoA2S (Table 1). This, however, appears to differ somewhat from an analysis of NMR data for the methyl aglycone of IdoA2S, which suggests the reverse preference. ADDIN REFMGR.CITE <Refman><Cite><Author>Hricov+?ni</Author><Year>2007</Year><RecNum>1185</RecNum><IDText>Relationship between structure and three-bond protonG??proton coupling constants in glycosaminoglycans</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1185</Ref_ID><Title_Primary>Relationship between structure and three-bond proton<f name="Symbol">G</f>Çôproton coupling constants in glycosaminoglycans</Title_Primary><Authors_Primary>Hricov+¡ni,M.</Authors_Primary><Authors_Primary>B+¡zik,F.</Authors_Primary><Date_Primary>2007</Date_Primary><Keywords>CONFORMATION</Keywords><Keywords>DFT</Keywords><Keywords>DFT calculations</Keywords><Keywords>GEOMETRIES</Keywords><Keywords>Glycosaminoglycans</Keywords><Keywords>Structure</Keywords><Keywords>theoretical calculation</Keywords><Keywords>Three-bond coupling constants</Keywords><Reprint>Not in File</Reprint><Start_Page>779</Start_Page><End_Page>783</End_Page><Periodical>CARBOHYDRATE RESEARCH</Periodical><Volume>342</Volume><Issue>6</Issue><Web_URL> name="System">CARBOHYDRATE RESEARCH</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>51 The msesMD simulation of IdoA2S predicts stabilisation of the two main skew-boat forms, 3S1 and 2SO (Figure 7a,b): on sulfation, the relative free energy of 2SO is lowered from 1.6 to 1.3 kcal/mol, although we note this 0.3 kcal/mol difference is towards the limit of estimate accuracy (Tables 1 and 2). For 3S1, the relative free energy is reduced from 1.8 to 1.1 kcal/mol on sulfation. As mentioned above, the 2SO conformation of the IdoA2S residue is significant, with NMR indicating this pucker is adopted by oligosaccharides that bind antithrombin III, including heparin.PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkd1ZXJyaW5pPC9BdXRob3I+PFllYXI+MjAxMzwvWWVhcj48
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ADDIN EN.CITE.DATA 50,52 On 2-O-sulfation of GlcA, the 4C1 chair is predicted to remain as the lowest energy pyranose conformer. This 4C1 preference is in accord with observation by NMR of GlcA2S residue pucker in heparan sulfate hexasaccharides. ADDIN REFMGR.CITE <Refman><Cite><Author>Hsieh</Author><Year>2014</Year><RecNum>1184</RecNum><IDText>Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1184</Ref_ID><Title_Primary>Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides</Title_Primary><Authors_Primary>Hsieh,Po Hung</Authors_Primary><Authors_Primary>Xu,Yongmei</Authors_Primary><Authors_Primary>Keire,David A.</Authors_Primary><Authors_Primary>Liu,Jian</Authors_Primary><Date_Primary>2014/4/25</Date_Primary><Keywords>ACID</Keywords><Keywords>AFFINITIES</Keywords><Keywords>AFFINITY</Keywords><Keywords>BINDING</Keywords><Keywords>BINDING AFFINITIES</Keywords><Keywords>binding affinity</Keywords><Keywords>BINDING-AFFINITY</Keywords><Keywords>SITE</Keywords><Keywords>SITES</Keywords><Keywords>STRUCTURAL-CHARACTERIZATION</Keywords><Keywords>Structure</Keywords><Reprint>Not in File</Reprint><Start_Page>681</Start_Page><End_Page>692</End_Page><Periodical>glycob</Periodical><Volume>24</Volume><Issue>8</Issue><Web_URL> name="System">GLYCOBIOLOGY</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">glycob</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>53 There is a smaller impact of sulfation on pucker distribution than for IdoA, both for the chair forms (Figure 6b) and for the intermediate boat/skew-boat populations (Figure 7c,d). For GlcA and GlcA2S, the 1S3 and BO,3 structures lie within ~2 kcal/mol of the lowest energy 4C1 conformation and are more favored than the 1C4 conformer (Tables 1 and 2). The substitution of the O2 hydroxyl group in GlcA with a bulkier O-sulfate in GlcA2S might be expected to lead to a decrease the 1C4 population, as the O2 group is in the axial orientation in the latter case. However the 0.6 kcal/mol increase in stability of the 1C4 pucker on sulfation appears to arise from the compensating presence in msesMD simulations of hydrogen bonding between the O4 hydroxyl of GlcA2S and its axial 2-O-sulfate group (data not shown).We next consider the hexosamine GlcNAc, a widely modified GAG monomer in nature. First, we consider the ?-anomer, and four of its commonly occurring variants (Figure 1): firstly, from N-deacetylation and subsequent N-sulfation of ?-GlcNAc, the N-sulfo glucosamine (?-GlcNS) is obtained. ?-GlcNS can undergo further O-sulfation through the action of heparan sulfate sulfotransferases at either the O3, O6 or both positions, leading to ?-GlcNS(3S), ?-GlcNS(6S) and ?-GlcNS(3S,6S) residues respectively (Figure 1a). For all five compounds, a similar free energy dependence on pucker angle ? is predicted by the 195 ns msesMD simulations (Figure 6c); these profiles indicate a distinct preference for the 4C1 chair form in all cases. This preference for the 4C1 conformation by ?-GlcNS agrees with previous combined MD and NMR analysis of ?-GlcNS conformation within a disaccharide and tetrasaccharide.PFJlZm1hbj48Q2l0ZT48QXV0aG9yPlNpbmdoPC9BdXRob3I+PFllYXI+MjAxNjwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 27,28 The ability of GlcNAc to sample non-chair conformations has been highlighted previously.PFJlZm1hbj48Q2l0ZT48QXV0aG9yPlRvcGluPC9BdXRob3I+PFllYXI+MjAxNjwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 23,54 Indeed, we find a range of puckers are sampled by GlcNAc in the msesMD simulations (Figure 7e-i). For example, for ?-GlcNAc, a preference for the 4C1 over 1C4 conformation is computed here as 1.4 kcal/mol (Table 2, Figure 7e); this energy difference was estimated as 3.5 kcal/mol from previous analysis of two 10 ?s MD simulations of ?-GlcNAc using the GLYCAM 06 force field. ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2011</Year><RecNum>1118</RecNum><IDText>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1118</Ref_ID><Title_Primary>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2011</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1651</Start_Page><End_Page>1662</End_Page><Periodical>glycob</Periodical><Volume>21</Volume><Issue>12</Issue><ZZ_JournalFull><f name="System">GLYCOBIOLOGY</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">glycob</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>28 Accessible from this 4C1 minimum are a range of boat/skew-boat forms at ? 90 via a barrier of ~8 kcal/mol (Figure 6c). Here, resolving the equatorial pseudorotation region obtained by msesMD simulation finds a diverse range of boat/skew-boat conformations (Figure 7e-i), with the most stable conformers occupying energies ~4.0 kcal/mol from the 4C1 minimum (Table 2). A similar range of boat and skew-boat conformers were obtained from the 2 x 10 ?s MD study. ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2011</Year><RecNum>1118</RecNum><IDText>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1118</Ref_ID><Title_Primary>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2011</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1651</Start_Page><End_Page>1662</End_Page><Periodical>glycob</Periodical><Volume>21</Volume><Issue>12</Issue><ZZ_JournalFull><f name="System">GLYCOBIOLOGY</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">glycob</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>28 Nevertheless, sampling the complex puckering landscape of sulfated ?-GlcNAc systems is challenging ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2012</Year><RecNum>1119</RecNum><IDText>Assigning kinetic 3D-signatures to glycocodes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1119</Ref_ID><Title_Primary>Assigning kinetic 3D-signatures to glycocodes</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2012</Date_Primary><Reprint>Not in File</Reprint><Start_Page>5843</Start_Page><End_Page>5848</End_Page><Periodical>Phys.Chem.Chem.Phys.</Periodical><Volume>14</Volume><Issue>16</Issue><ZZ_JournalStdAbbrev><f name="System">Phys.Chem.Chem.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Sattelle</Author><Year>2011</Year><RecNum>1118</RecNum><IDText>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1118</Ref_ID><Title_Primary>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2011</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1651</Start_Page><End_Page>1662</End_Page><Periodical>glycob</Periodical><Volume>21</Volume><Issue>12</Issue><ZZ_JournalFull><f name="System">GLYCOBIOLOGY</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">glycob</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>28,55 and in that same study, 2 x 10 ?s MD simulations of ?-GlcNS(3S) and ?-GlcNS(3S,6S) observed no 4C1-to-1C4 transitions, and for the ?-GlcNS(6S) residue, one transition was observed. ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2011</Year><RecNum>1118</RecNum><IDText>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1118</Ref_ID><Title_Primary>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2011</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1651</Start_Page><End_Page>1662</End_Page><Periodical>glycob</Periodical><Volume>21</Volume><Issue>12</Issue><ZZ_JournalFull><f name="System">GLYCOBIOLOGY</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">glycob</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>28 Here, we observe frequent sampling over the pucker coordinate, leading to computation of a continuous puckering free energy surface across ??(Figure 6c); we do note, however, that not all replicas of the swarm sampled the complete range of ? (Figure S19). We also compute the pucker free energy profiles of the ?-anomer of GlcNAc. As for the ?-anomer, there is a preference for the 4C1 chair conformer; the 1C4 conformer is predicted to be 3.8 kcal/mol less stable, as compared to 1.4 kcal/mol for the ??anomer (Figure 6d, Table 2). The energy barrier to accessing the 1C4 state is also larger. This could be explained by the energetic penalty of the additional axial substituent (the O1 hydroxyl group) in this ?-GlcNAc conformer. For the ?-anomer, there a range of low energy non-chair conformers (Figure 7j, Table 2). Sulfation at the 6-position of ?-GlcNAc is predicted by msesMD simulation to stabilize the 1C4 chair pucker by 0.8 kcal/mol (Figure 6d, Table 2). The ?? profile closely resembles that of ?-GlcNAc (Figure 7j,k) with a broadly similar distribution of puckers (Table 2). Finally, we consider the ring pucker of hexosamine, ?-GalNAc, and three of its derivatives found in GAGs: these are sulfated at O4, O6 or both these positions in ?-GalNAc, and are denoted ?-GalNAc(4S), ?-GalNAc(6S) and ?-GalNAc(4S,6S) respectively (Figure 1). Relatively little work has been performed computationally or experimentally on the study of ?-GalNAc ring pucker, ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2012</Year><RecNum>1119</RecNum><IDText>Assigning kinetic 3D-signatures to glycocodes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1119</Ref_ID><Title_Primary>Assigning kinetic 3D-signatures to glycocodes</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2012</Date_Primary><Reprint>Not in File</Reprint><Start_Page>5843</Start_Page><End_Page>5848</End_Page><Periodical>Phys.Chem.Chem.Phys.</Periodical><Volume>14</Volume><Issue>16</Issue><ZZ_JournalStdAbbrev><f name="System">Phys.Chem.Chem.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>55 and to our knowledge, no work exists on the effect of O-sulfation on its pucker. As for GlcNAc and its derivatives, we find here that msesMD simulation of the four ?-GalNAc-based monosaccharides exhibit a strong preference for the 4C1 chair conformer (Figure 6e). The predicted barrier to accessing the boat/skew-boat region is also similar to that of ?-GlcNAc, with a value of ~7 kcal/mol (Figure 6e). Here, however, the comparison with GlcNAc ends: this boat/skew-boat region of ?-GalNAc and its sulfated forms appears restricted to the 1S3 skew-boat pucker (Figure 8a-d), as opposed to the more diverse range of structures accessible to GlcNAc and its derivatives (Figure 7e-k). Based on the msesMD simulations, the computed stability of the 1S3 form is between 3.5 and 4.4 kcal/mol lower than that of the 4C1 minimum of ?-GalNAc and its derivatives (Table 2). Most striking however is the lack of stability of its 1C4 form. Indeed, the lowest energy 1C4 structures are found for ?-GalNAc(4S) and ?-GalNAc(4S,6S), which lie 5.0 and 8.3 kcal/mol above the 4C1 form respectively. A previous 5 ?s MD simulation of ?-GalNAc in explicit aqueous solvent ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2012</Year><RecNum>1119</RecNum><IDText>Assigning kinetic 3D-signatures to glycocodes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1119</Ref_ID><Title_Primary>Assigning kinetic 3D-signatures to glycocodes</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2012</Date_Primary><Reprint>Not in File</Reprint><Start_Page>5843</Start_Page><End_Page>5848</End_Page><Periodical>Phys.Chem.Chem.Phys.</Periodical><Volume>14</Volume><Issue>16</Issue><ZZ_JournalStdAbbrev><f name="System">Phys.Chem.Chem.Phys.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>55 observed the inaccessibility of the 1C4 form, where no conformer was sampled over the duration of these simulations. Similarly, no 1C4 conformer is sampled for ?-GalNAc and ?-GalNAc(6S) by msesMD simulations here, although it is sampled in the ?-GalNAc(4S) and ?-GalNAc(4S,6S) sulfated forms (Figure S21). In terms of the physical origin of these observations, we note that a 4C1-to-1C4 transition for ?-GalNAc involves displacing four of its ring substituents from equatorial to axial positions, including the bulky N-acetyl group; this leads to unfavorable 1,3-diaxial interactions in the 1C4 structure of ?-GalNAc. The effect of the N-acetyl group can be evidenced by comparing the pucker free energy profile for ?-GalNAc with that computed for ?-Gal (Figure 6e,f). The 1C4 conformation is stabilised significantly in the absence of the N-acetyl group (Figure 6f), such that it lies 7.4 kcal/mol above the 4C1 state. This compares with an inferred energy preference for 4C1 of 5.2 kcal/mol from the NMR analysis of ?-Gal by Angyal. ADDIN REFMGR.CITE <Refman><Cite><Author>Angyal</Author><Year>1968</Year><RecNum>1186</RecNum><IDText>Conformational analysis in carbohydrate chemistry. I. Conformational free energies. The conformations and &#945; : &#946; ratios of aldopyranoses in aqueous solution</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1186</Ref_ID><Title_Primary>Conformational analysis in carbohydrate chemistry. I. Conformational free energies. The conformations and &#945; : &#946; ratios of aldopyranoses in aqueous solution</Title_Primary><Authors_Primary>Angyal,S.J.</Authors_Primary><Date_Primary>1968</Date_Primary><Keywords>analysis</Keywords><Keywords>AQUEOUS-SOLUTION</Keywords><Keywords>carbohydrate</Keywords><Keywords>CONFORMATION</Keywords><Keywords>conformational analysis</Keywords><Keywords>CONFORMATIONAL-ANALYSIS</Keywords><Keywords>CONFORMATIONS</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Keywords>INTERACTION ENERGIES</Keywords><Reprint>Not in File</Reprint><Start_Page>2737</Start_Page><End_Page>2746</End_Page><Periodical>Australian Journal of Chemistry</Periodical><Volume>21</Volume><Issue>11</Issue><Web_URL> name="System">Australian Journal of Chemistry</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>48 In both ?-GalNAc and ?-Gal, introduction of sulfation at the O6 position only compounds this effect, further destabilising the 1C4 ring pucker (Figure 6e,f). Conversely, msesMD simulation predicts that O4 sulfation of ?-GalNAc significantly stabilises its 1C4 pucker, such that it lies at 5 kcal/mol above the 4C1 chair (Table 2). This is in part due to the bulky O4 sulfate group occupying a strained axial position in the 4C1 chair, thus shifting the conformational equilibrium towards 1C4. The 1C4 form is also stabilised by formation of intra-ring hydrogen bonding between the sulfate and hydroxyls groups in ?-GalNS(4S) (Figure 9a); for ?-GalNS(4S,6S), intra-ring hydrogen bonding is also observed (Figure 9b). However, for this monosaccharide, minimizing electrostatic repulsion between sulfate groups is an additional factor governing ring conformation. In this case, one might expect the presence of increased salt to alleviate electrostatic repulsion within ?-GalNAc(4S,6S). To examine the effect of salt, we performed comparative msesMD simulations of ?-GalNAc, ?-GalNAc(4S), ?-GalNAc(6S) and ?-GalNAc(4S,6S) in the presence of additional NaCl at a concentration of ~150-175 mM. For the neutral ?-GalNAc monosaccharide and the monoanionic ?-GalNAc(6S), the pucker free energy profiles are identical to within error with the msesMD simulations at zero ionic strength (Figures S23 and S24). For ?-GalNAc(4S), there is a small ~ 0.6 kcal/mol destabilisation of the 1C4 conformer in the presence of salt, such that it lies 6 kcal/mol above the 4C1 conformer; we note, however, that this computed change in stability lies towards the limit in convergence of the estimated free energy for ?-GalNAc(4S) via msesMD (Figures S25 and S28). As anticipated due to increased screening of intramolecular charge repulsion within the dianionic ?-GalNAc(4S,6S), there is a larger influence of salt on this monosaccharide: the 1C4 conformer is stabilised by ~ 2 kcal/mol (Figures S23 and S24), such that it lies 6 kcal/mol above the 4C1 conformer, as is the case for ?-GalNAc(4S). Therefore, we predict that the influence of 6-sulfation is largely negated by salt effects; however, the impact of the 4-sulfation on stabilising the 1C4 pucker of ?-GalNAc remains. 4. ConclusionsIn this work, we explore the ring puckering landscape of a range of glycosaminoglycan-related monosaccharides using enhanced sampling molecular dynamics simulations via the msesMD method. We first demonstrate that msesMD efficiently probes the thermodynamics of pyranose rings for four monosaccharides. At an order of magnitude lower computational cost, the application of a swarm biasing potential to two ring dihedrals via msesMD yielded puckering free energy profiles for the four monosaccharides in quantitative agreement with long timescale MD simulations. In the cases of ??Glc and GlcA, unbiased MD simulations over 15 ?s in length were required for adequate sampling; thus, some monosaccharides appear to require longer simulation times than previously thought. ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2011</Year><RecNum>1118</RecNum><IDText>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1118</Ref_ID><Title_Primary>Is N-acetyl-D-glucosamine a rigid 4C1 chair?</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2011</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1651</Start_Page><End_Page>1662</End_Page><Periodical>glycob</Periodical><Volume>21</Volume><Issue>12</Issue><ZZ_JournalFull><f name="System">GLYCOBIOLOGY</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">glycob</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Sattelle</Author><Year>2010</Year><RecNum>1113</RecNum><IDText>Free energy landscapes of iduronic acid and related monosaccharides</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1113</Ref_ID><Title_Primary>Free energy landscapes of iduronic acid and related monosaccharides</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Hansen,Steen U.</Authors_Primary><Authors_Primary>Gardiner,John</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>ACID</Keywords><Keywords>ENERGIES</Keywords><Keywords>ENERGY</Keywords><Keywords>FREE ENERGY</Keywords><Keywords>FREE-ENERGIES</Keywords><Keywords>FREE-ENERGY</Keywords><Reprint>Not in File</Reprint><Start_Page>13132</Start_Page><End_Page>13134</End_Page><Periodical>J.Am.Chem.Soc.</Periodical><Volume>132</Volume><Issue>38</Issue><ZZ_JournalStdAbbrev><f name="System">J.Am.Chem.Soc.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>12,28 For the msesMD simulations, we also note that the parameters for the swarm biasing potential employed in this work are the same as used in previous efficient sampling of glycosidic and peptide backbone torsional degrees of freedom, ADDIN REFMGR.CITE <Refman><Cite><Author>Alibay</Author><Year>2018</Year><RecNum>1121</RecNum><IDText>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1121</Ref_ID><Title_Primary>Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics</Title_Primary><Authors_Primary>Alibay,Irfan</Authors_Primary><Authors_Primary>Burusco,Kepa K.</Authors_Primary><Authors_Primary>Bruce,Neil J.</Authors_Primary><Authors_Primary>Bryce,Richard A.</Authors_Primary><Date_Primary>2018/3/8</Date_Primary><Keywords>AQUEOUS-SOLUTION</Keywords><Keywords>DYNAMICS</Keywords><Keywords>IDENTIFICATION</Keywords><Keywords>molecular dynamics</Keywords><Keywords>MOLECULAR-DYNAMICS</Keywords><Reprint>Not in File</Reprint><Start_Page>2462</Start_Page><End_Page>2474</End_Page><Periodical>J.Phys.Chem.B</Periodical><Volume>122</Volume><Issue>9</Issue><Web_URL> name="System">J.Phys.Chem.B</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>23 suggesting a degree of transferability in this biasing potential. We then applied msesMD simulations to compare the puckering free energy landscapes for a range of glycosaminoglycan-related monosaccharides: we find that for sulfated and unsulfated forms of IdoA, GlcA, GlcNAc and, to a lesser degree, GalNAc, the free energy profiles are rather similar. For IdoA and IdoA2S, an NMR analysis of eight heparin sulfate-based hexasaccharides concluded that the variation in 4C1:2SO:1C4 populations of the IdoA/IdoA2S residue was dictated by the differing sulfation patterns of neighbouring residues. ADDIN REFMGR.CITE <Refman><Cite><Author>Hsieh</Author><Year>2016</Year><RecNum>1115</RecNum><IDText>Uncovering the relationship between sulphation patterns and conformation of iduronic acid in heparan sulphate</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1115</Ref_ID><Title_Primary>Uncovering the relationship between sulphation patterns and conformation of iduronic acid in heparan sulphate</Title_Primary><Authors_Primary>Hsieh,Po Hung</Authors_Primary><Authors_Primary>Thieker,David F.</Authors_Primary><Authors_Primary>Guerrini,Marco</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Authors_Primary>Liu,Jian</Authors_Primary><Date_Primary>2016</Date_Primary><Keywords>ACID</Keywords><Keywords>CONFORMATION</Keywords><Reprint>Not in File</Reprint><Start_Page>29602</Start_Page><Periodical>Sci.Rep.</Periodical><Volume>6</Volume><ZZ_JournalStdAbbrev><f name="System">Sci.Rep.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>29 This suggests that inter-residue hydrogen bonds and other interactions arising from the interplay of sulfated and non-sulfated groups play a key role in dictating the shape and function of GAG polysaccharides.Secondly, our msesMD study of ?-GalNAc derivatives found that, although the galactosamine ring was more rigid than for GlcNAc, 4-O-sulfation of ?-GalNAc led to a somewhat unexpected stabilisation of the 1C4 form and lowering of the energy barrier leading to this conformer (Figure 6d). Although still predicted as 5 – 6 kcal/mol higher in energy than the 4C1 conformer, the potential access to a 1C4 form could be relevant to the structure, interaction and function of polysaccharides such as dermatan sulfate. The anticoagulant activity of dermatan sulfate from Ascidian nigra, possessing 6-O-sulfated ?-GalNAc residues, has been examined; ADDIN REFMGR.CITE <Refman><Cite><Author>Pavao</Author><Year>1995</Year><RecNum>1122</RecNum><IDText>A unique dermatan sulfate-like glycosaminoglycan from ascidian: its structure and the effect of its unusual sulfation pattern on anticoagulant activity</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1122</Ref_ID><Title_Primary>A unique dermatan sulfate-like glycosaminoglycan from ascidian: its structure and the effect of its unusual sulfation pattern on anticoagulant activity</Title_Primary><Authors_Primary>Pavao,Mauro SG</Authors_Primary><Authors_Primary>Mourao,Paulo AS</Authors_Primary><Authors_Primary>Mulloy,Barbara</Authors_Primary><Authors_Primary>Tollefsen,Douglas M.</Authors_Primary><Date_Primary>1995</Date_Primary><Keywords>Structure</Keywords><Reprint>Not in File</Reprint><Start_Page>31027</Start_Page><End_Page>31036</End_Page><Periodical>J.Biol.Chem.</Periodical><Volume>270</Volume><Issue>52</Issue><ZZ_JournalStdAbbrev><f name="System">J.Biol.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>56 this was compared with the activity of the mammalian form, which contains solely 4-O-sulfated ?-GalNAc. It was found that only the mammalian form exhibited anticoagulant activity and potent interaction with heparin cofactor II. This suggests that 4-O-sulfation of ?-GalNAc residues is required for dermatan sulfate’s anticoagulant function. The increased flexibility of the ?-GalNAc(4S) ring predicted here may play a role in this, although the overall conformation and activity of these glycosaminoglycans is likely due to a complex sum of saccharide intra- and interresidue structure, ADDIN REFMGR.CITE <Refman><Cite><Author>Sattelle</Author><Year>2010</Year><RecNum>1188</RecNum><IDText>A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1188</Ref_ID><Title_Primary>A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation</Title_Primary><Authors_Primary>Sattelle,Benedict M.</Authors_Primary><Authors_Primary>Shakeri,Javad</Authors_Primary><Authors_Primary>Roberts,Ian S.</Authors_Primary><Authors_Primary>Almond,Andrew</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>CONFORMATION</Keywords><Keywords>MODEL</Keywords><Reprint>Not in File</Reprint><Start_Page>291</Start_Page><End_Page>302</End_Page><Periodical>CARBOHYDRATE RESEARCH</Periodical><Volume>345</Volume><Issue>2</Issue><ZZ_JournalFull><f name="System">CARBOHYDRATE RESEARCH</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>57 complementing interactions with heparin cofactor II. ADDIN REFMGR.CITE <Refman><Cite><Author>Pavao</Author><Year>1995</Year><RecNum>1122</RecNum><IDText>A unique dermatan sulfate-like glycosaminoglycan from ascidian: its structure and the effect of its unusual sulfation pattern on anticoagulant activity</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1122</Ref_ID><Title_Primary>A unique dermatan sulfate-like glycosaminoglycan from ascidian: its structure and the effect of its unusual sulfation pattern on anticoagulant activity</Title_Primary><Authors_Primary>Pavao,Mauro SG</Authors_Primary><Authors_Primary>Mourao,Paulo AS</Authors_Primary><Authors_Primary>Mulloy,Barbara</Authors_Primary><Authors_Primary>Tollefsen,Douglas M.</Authors_Primary><Date_Primary>1995</Date_Primary><Keywords>Structure</Keywords><Reprint>Not in File</Reprint><Start_Page>31027</Start_Page><End_Page>31036</End_Page><Periodical>J.Biol.Chem.</Periodical><Volume>270</Volume><Issue>52</Issue><ZZ_JournalStdAbbrev><f name="System">J.Biol.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>56 Work on elucidating the role of geometric conformation in GAGs and how it is encoded by selective chain decoration is still in its early stages. ADDIN REFMGR.CITE <Refman><Cite><Author>Rudd</Author><Year>2010</Year><RecNum>1157</RecNum><IDText>The conformation and structure of GAGs: recent progress and perspectives</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1157</Ref_ID><Title_Primary>The conformation and structure of GAGs: recent progress and perspectives</Title_Primary><Authors_Primary>Rudd,T.R.</Authors_Primary><Authors_Primary>Skidmore,M.A.</Authors_Primary><Authors_Primary>Guerrini,M.</Authors_Primary><Authors_Primary>Hricovini,M.</Authors_Primary><Authors_Primary>Powell,A.K.</Authors_Primary><Authors_Primary>Siligardi,G.</Authors_Primary><Authors_Primary>Yates,E.A.</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>CONFORMATION</Keywords><Keywords>Structure</Keywords><Reprint>Not in File</Reprint><Start_Page>567</Start_Page><End_Page>574</End_Page><Periodical>Curr.Opin.Struct.Biol.</Periodical><Volume>20</Volume><Issue>5</Issue><ZZ_JournalStdAbbrev><f name="System">Curr.Opin.Struct.Biol.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>5 This is in part due to the synthetic challenges of making point modified polysaccharides ADDIN REFMGR.CITE <Refman><Cite><Author>Jayson</Author><Year>2014</Year><RecNum>1141</RecNum><IDText>The development of anti-angiogenic heparan sulfate oligosaccharides</IDText><MDL Ref_Type="Generic"><Ref_Type>Generic</Ref_Type><Ref_ID>1141</Ref_ID><Title_Primary>The development of anti-angiogenic heparan sulfate oligosaccharides</Title_Primary><Authors_Primary>Jayson,Gordon C.</Authors_Primary><Authors_Primary>Miller,Gavin J.</Authors_Primary><Authors_Primary>Hansen,Steen U.</Authors_Primary><Authors_Primary>Barath,Marek</Authors_Primary><Authors_Primary>Gardiner,John M.</Authors_Primary><Authors_Primary>Avizienyte,Egle</Authors_Primary><Date_Primary>2014</Date_Primary><Reprint>Not in File</Reprint><Publisher>Portland Press Limited</Publisher><ISSN_ISBN>0300-5127</ISSN_ISBN><ZZ_WorkformID>33</ZZ_WorkformID></MDL></Cite><Cite><Author>Hansen</Author><Year>2012</Year><RecNum>1158</RecNum><IDText>First gram-scale synthesis of a heparin-related dodecasaccharide</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1158</Ref_ID><Title_Primary>First gram-scale synthesis of a heparin-related dodecasaccharide</Title_Primary><Authors_Primary>Hansen,Steen U.</Authors_Primary><Authors_Primary>Miller,Gavin J.</Authors_Primary><Authors_Primary>Jayson,Gordon C.</Authors_Primary><Authors_Primary>Gardiner,John M.</Authors_Primary><Date_Primary>2012</Date_Primary><Reprint>Not in File</Reprint><Start_Page>88</Start_Page><End_Page>91</End_Page><Periodical>Org.Lett.</Periodical><Volume>15</Volume><Issue>1</Issue><ZZ_JournalStdAbbrev><f name="System">Org.Lett.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>58,59 and in part because of the difficulty in accurately simulating these highly charged compounds, both in terms of sampling its complex conformational landscape and in capturing its physical behaviour via a classical potential energy function.PFJlZm1hbj48Q2l0ZT48QXV0aG9yPlNpbmdoPC9BdXRob3I+PFllYXI+MjAxNjwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 5,13,27 In regard to this last point, the simulation study here employed the GLYCAM06 force field ADDIN REFMGR.CITE <Refman><Cite><Author>Kirschner</Author><Year>2008</Year><RecNum>1143</RecNum><IDText>GLYCAM06: a generalizable biomolecular force field. Carbohydrates</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1143</Ref_ID><Title_Primary>GLYCAM06: a generalizable biomolecular force field. Carbohydrates</Title_Primary><Authors_Primary>Kirschner,Karl N.</Authors_Primary><Authors_Primary>Yongye,Austin B.</Authors_Primary><Authors_Primary>Tschampel,Sarah M.</Authors_Primary><Authors_Primary>Gonz+ílez<f name="Symbol">G</f>ÇÉOuteiri+¦o,Jorge</Authors_Primary><Authors_Primary>Daniels,Charlisa R.</Authors_Primary><Authors_Primary>Foley,B.Lachele</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>FORCE FIELD</Keywords><Keywords>FORCE-FIELD</Keywords><Keywords>GLYCAM06</Keywords><Reprint>Not in File</Reprint><Start_Page>622</Start_Page><End_Page>655</End_Page><Periodical>put.Chem.</Periodical><Volume>29</Volume><Issue>4</Issue><ZZ_JournalStdAbbrev><f name="System">put.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>26 and its recently introduced extension for modelling GAGs. ADDIN REFMGR.CITE <Refman><Cite><Author>Singh</Author><Year>2016</Year><RecNum>1142</RecNum><IDText>Extension and validation of the GLYCAM force field parameters for modeling glycosaminoglycans</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1142</Ref_ID><Title_Primary>Extension and validation of the GLYCAM force field parameters for modeling glycosaminoglycans</Title_Primary><Authors_Primary>Singh,Arunima</Authors_Primary><Authors_Primary>Tessier,Matthew B.</Authors_Primary><Authors_Primary>Pederson,Kari</Authors_Primary><Authors_Primary>Wang,Xiaocong</Authors_Primary><Authors_Primary>Venot,Andre P.</Authors_Primary><Authors_Primary>Boons,Geert Jan</Authors_Primary><Authors_Primary>Prestegard,James H.</Authors_Primary><Authors_Primary>Woods,Robert J.</Authors_Primary><Date_Primary>2016/2/9</Date_Primary><Keywords>FORCE FIELD</Keywords><Keywords>FORCE-FIELD</Keywords><Keywords>PARAMETERS</Keywords><Keywords>VALIDATION</Keywords><Reprint>Not in File</Reprint><Start_Page>927</Start_Page><End_Page>935</End_Page><Periodical>Can.J.Chem.</Periodical><Volume>94</Volume><Issue>11</Issue><Web_URL> name="System">Canadian Journal of Chemistry</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Can.J.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>27 Puckering information was not directly used in the fitting of the force field but was used in its validation. In general, the converged results we obtain from MD and msesMD give reasonable agreement with NMR and other simulation studies. We note, however, from MD and msesMD simulations that the 1C4 conformer of ?-Glc appears to be overstabilized relative to its population inferred from NMR experiments. Similarly, the favoured pucker state of IdoA2S appears at variance with that obtained from NMR of its methyl agylcone. For modelling the complex intra- and intermolecular physics of neutral and charged saccharide molecules in solution, it may be that moving beyond a fixed point charge force field could prove beneficial, as has been found elsewhere.PFJlZm1hbj48Q2l0ZT48QXV0aG9yPlBhdGVsPC9BdXRob3I+PFllYXI+MjAxNDwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 60,61 However, for a given force field, the use of the msesMD enhanced sampling MD method provides an intuitive and efficient approach for sampling the puckering free energy landscape. As models of GAG monosaccharides are further validated and refined, simulation-based approaches will afford a useful aid to understanding the structure and interactions of their oligomeric and polymeric forms.Supporting Information. Structural, energetic and error analyses of MD simulations of GAG-related monosaccharides. Microsoft excel file with stochastic transition matrices (Supplemental Data). This material is available free of charge via the Internet at thank Kepa Burusco and Rocco Meli for helpful discussions. This project made use of time granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (), supported by EPSRC (grant no. EP/L000253/1). The authors would also like to acknowledge the use of the Computational Shared Facility at the University of Manchester. ADDIN REFMGR.REFLIST References(1) Dwek, R. A. Glycobiology: Toward Understanding the Function of Sugars. Chem. Rev. 1996, 96, 683-720.(2) Jackson, R. L.; Busch, S. J.; Cardin, A. D. Glycosaminoglycans: Molecular Properties, Protein Interactions, and Role in Physiological Processes. Physiolog. Rev. 1991, 71, 481-539.(3) Bishop, J. R.; Schuksz, M.; Esko, J. D. Heparan Sulphate Proteoglycans Fine-Tune Mammalian Physiology. Nature 2007, 446, 1030.(4) Gama, C. I.; Tully, S. E.; Sotogaku, N.; Clark, P. M.; Rawat, M.; Vaidehi, N.; Goddard III, W. A.; Nishi, A.; Hsieh-Wilson, L. C. Sulfation Patterns of Glycosaminoglycans Encode Molecular Recognition and Activity. Nature Chem. Biol. 2006, 2, 467.(5) Rudd, T. R.; Skidmore, M. A.; Guerrini, M.; Hricovini, M.; Powell, A. K.; Siligardi, G.; Yates, E. A. The Conformation and Structure of GAGs: Recent Progress and Perspectives. 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Glycobiology 2014, 24, 681-692.(54) Topin, J.; Lelimousin, M.; Arnaud, J.; Audfray, A.; Perez, S.; Varrot, A.; Imberty, A. The Hidden Conformation of Lewis X, a Human Histo-Blood Group Antigen, Is a Determinant for Recognition by Pathogen Lectins. ACS Chem. Biol. 2016, 11, 2011-2020.(55) Sattelle, B. M.; Almond, A. Assigning Kinetic 3D-Signatures to Glycocodes. Phys. Chem. Chem. Phys. 2012, 14, 5843-5848.(56) Pavao, M. S.; Mourao, P. A.; Mulloy, B.; Tollefsen, D. M. A Unique Dermatan Sulfate-Like Glycosaminoglycan From Ascidian: Its Structure and the Effect of Its Unusual Sulfation Pattern on Anticoagulant Activity. J. Biol. Chem. 1995, 270, 31027-31036.(57) Sattelle, B. M.; Shakeri, J.; Roberts, I. S.; Almond, A. A 3D-Structural Model of Unsulfated Chondroitin From High-Field NMR: 4-Sulfation Has Little Effect on Backbone Conformation. Carbohydr. Res. 2010, 345, 291-302.(58) Jayson, G. C.; Miller, G. J.; Hansen, S. U.; Barath, M.; Gardiner, J. M.; Avizienyte, E. The development of anti-angiogenic heparan sulfate oligosaccharides. 2014. Portland Press Limited. (59) Hansen, S. U.; Miller, G. J.; Jayson, G. C.; Gardiner, J. M. First Gram-Scale Synthesis of a Heparin-Related Dodecasaccharide. Org. Lett. 2012, 15, 88-91.(60) Patel, D. S.; He, X.; MacKerell Jr, A. D. Polarizable Empirical Force Field for Hexopyranose Monosaccharides Based on the Classical Drude Oscillator. J. Phys. Chem. B 2014, 119, 637-652.(61) Pandey, P.; Aytenfisu, A. H.; MacKerell Jr, A. D.; Mallajosyula, S. S. Drude Polarizable Force Field Parametrization of Carboxylate and N-Acetyl Amine Carbohydrate Derivatives. J. Chem. Theory Comput. 2019, 15, 4982-5000. Table 1 Relative free energy of 1C4 and selected non-chair states of monosaccharides relative to 4C1 chair form (kcal/mol), computed via MD and 195 ns msesMD simulations. Non-chair conformers (energies in parentheses) selected within 0.5 kcal/mol of the lowest energy non-chair conformer.Table 2 Relative free energy of 1C4 and selected non-chair conformers of monosaccharides relative to 4C1 chair form (kcal/mol), computed via 195 ns msesMD simulations. Non-chair conformers selected within 0.5 kcal/mol of the lowest energy non-chair conformer.9525006350(a)020000(a)10922005080(b)020000(b)Figure 1 (a) Monosaccharides α-D-glucose (α-Glc), β-D-glucose (β-Glc), α-L-iduronic acid (α-Glc), β-D-glucuronic acid (β-GlcA); ?- and β-GlcNAc, β-GalNAc, β-Gal and their sulfated variants. (b) Schematic diagram of location of selected pyranose ring conformers on Cremer-Pople (?,?) surface. Figure 2 Free energy profiles as a function of Cremer-Pople puckering angle θ for (a) α-Glc, (b) β-Glc, (c) IdoA, and (d) GlcA calculated via unbiased MD trajectories of varying length (colored points) or via 195 ns msesMD simulations (black circle). Energies in kcal/mol. Regions corresponding to chair (C), envelope or half-chair (E/HC) and boat or skew-boat (B/SB) ring conformations are indicated.Figure 3 Time series of Cremer-Pople θ angle computed from respective 20 ?s MD and 195 ns msesMD simulations of (a,b) α-Glc, (c,d) β-Glc, (e,f) IdoA, and (g,h) GlcA. For msesMD simulations, the θ angle values are coloured according to replica. Figure 4 Evaluation of the convergence of relative free energy profiles computed via msesMD simulations of duration 45 ns (black), 95 ns (dark blue), 145 ns (green), and 195 ns (light green), as a function of Cremer-Pople puckering angle θ for (a) α-Glc, (b) β-Glc, (c) IdoA and (d) GlcA. Energies in kcal/mol. Regions corresponding to chair (C), envelope or half-chair (E/HC) and boat or skew-boat (B/SB) ring conformations are indicated.Figure 5 Relative free energy profiles as a function of Cremer-Pople puckering angles θ? computed from respective 20 ?s MD and 195 ns msesMD simulations of (a,b) α-Glc, (c,d) β-Glc, (e,f) α-IdoA and (g,h) β-GlcA. Regions corresponding to chair (C), boat (B), and skew-boat (S) ring conformations are indicated. Displayed relative free energy scale ranges from 0 kcal/mol (black) to 8 kcal/mol (white). Unsampled regions have been given a value of 8 kcal/mol.Figure 6 Relative free energy profiles as a function of Cremer-Pople angle θ evaluating the impact of ring modification of (a) ?-IdoA, (b) ?-GlcA, (c) ?-GlcNAc, (d) ?-GlcNAc, (e) ?-GalNAc and (f) ?-Gal, computed from 195 ns msesMD simulations. Energies in kcal/mol. Regions corresponding to chair (C), envelope or half-chair (E/HC) and boat or skew-boat (B/SB) ring conformations are indicated.Figure 7 Relative free energy profiles as a function of Cremer-Pople puckering angles θ? from 195 ns msesMD simulation of (a) ?-IdoA, (b) ?-IdoA2S, (c) ?-GlcA, (d) ?-GlcA2S, (e) ?-GlcNAc, (f) ?-GlcNS, (g) ??GlcNS(3S), (h) ??GlcNAc(6S), (i) ??GlcNAc(3S,6S), (j) ?-GlcNAc, and (k) ?-GlcNAc(6S). Regions corresponding to chair (C), boat (B) and skew-boat (S) ring conformations are indicated. Displayed relative free energy scale ranges from 0 kcal/mol (black) to 8 kcal/mol (white). Unsampled regions have been given a value of 8 kcal/mol.Figure 8 Relative free energy profiles as a function of Cremer-Pople puckering angles θ? from 195 ns msesMD simulation of (a) ?-GalNAc, (b) ?-GalNAc(4S), (c) ?-GalNAc(6S), (d) ?-GalNAc(4S,6S), (e) ?-Gal and (f) ?-Gal(6S). Regions corresponding to chair (C), boat (B) and skew-boat (S) ring conformations are indicated. Displayed relative free energy scale ranges from 0 kcal/mol (black) to 8 kcal/mol (white). Unsampled regions have been given a value of 8 kcal/mol.Figure 9 Selected conformations of 1C4 conformers of (a) ?-GalNAc(4S) and (b) ?-GalNAc(4S,6S) indicating the type of intra-molecular hydrogen bond pattern which can be accessed (with representative distances in ?). Atoms are annotated by colour for; carbon (green), oxygen (red), nitrogen (blue), sulfur (yellow), and hydrogen (white).TOC graphic ................
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