inhibiting 
 or co - opting these interactions could lead to new classes of therapeutics , but despite a few notable successes , harnessing and controlling these interactions remains challenging . 
 to elucidate and intervene in the biological processes mediated by 
 protein  carbohydrate interactions , nonetheless , the precise nature 
 and balance of forces that drive the complexation of carbohydrates 
 by proteins are not fully understood . the importance of hydrogen 
 bonds between the carbohydrate hydroxyl 
 groups and polar moieties of amino acids in the binding of carbohydrates 
 by proteins is well recognized . however , the role played by hydrophobic 
 aliphatic and aromatic side chains in binding water - soluble carbohydrates 
 is more obscure , with emphasis placed on interactions with carbohydrate 
 c  h groups through the hydrophobic effect . carbohydrate - aromatic interactions 
 are increasingly the subject of study in their own right , and an underlying contributer to affinity is 
 the ch interaction , i.e. , the interaction of an aromatic 
 -system with a c  h bond . indeed , carbohydrate aromatic 
 interactions have been examined in model systems using a variety of 
 methods , including computational studies ; investigation of the folding 
 of synthetic glycopeptides designed to form intramolecular interactions ; 
 and the interrogation of small - molecule systems by solution - phase 
 nmr studies . aromatic 
 interactions , but some gaps in knowledge remain : the relative propensities 
 of specific monosaccharides and aromatic residues to participate in 
 carbohydrate  aromatic interactions have not been quantified , 
 nor is it known whether certain carbohydrate c carbohydrate 
 complexes , and it would facilitate the design of efficacious inhibitors . 
 answering these questions ch interactions 
 have an agreed dispersion , or van der waals component . however , additional 
 electrostatic contributions  namely , potentially attractive 
 interactions between partial positive charges on c  h protons 
 and the electronegative -system  are less certain . therefore , the importance of electronic effects in the species  i.e . , 
 the factors affecting these charges , such as inductive and stereoelectronic 
 effects  is not established . aromatic complexes have found cases both 
 where electronics are important for ch interactions , and where they do not play a 
 major role . to date , 
 such analyses have been restricted to specific protein families or 
 carbohydrate residues . thus , there is not yet a general 
 understanding of how the structural properties of individual monosaccharides 
 lead to their binding and discrimination through the inherent characteristics 
 and positioning of amino acids within carbohydrate - binding sites in 
 proteins . the increased size of the protein data bank ( pdb ) over the 
 past decade provides a rich source of 
 structural data on protein  carbohydrate complexes . we reasoned that quantitative analyses across 
 all protein classes would uncover general and clear principles of 
 protein  carbohydrate interactions , should they exist . our analyses reveal that the noncovalently bound carbohydrates 
 make more - numerous and more - specific contacts with protein side chains 
 than do covalently attached carbohydrates ( i.e. , in glycoproteins ) 
 in the pdb . in the binding sites of the former , polar amino acids 
 mostly occur with frequencies expected by chance ; aliphatic hydrophobic 
 residues are underrepresented , whereas electron - rich aromatic side 
 chains , particularly tryptophan , are favored . moreover , there are 
 preferred relative orientations of the aromatic and carbohydrate rings , 
 which depend on the identity of the saccharide residue . interactions to the electronegative aromatic rings are observed more 
 frequently for more - electropositive c h bonds , indicating important 
 contributions from both orbital overlap and complementary electronics 
 between the carbohydrate and -system . this analysis is supported 
 by determination of linear free energy relationships using substituted 
 indoles and methyl glycosides , which highlight a key role for electronic 
 effects in ch interactions . to generate the protein  carbohydrate 
 interaction database , 
 context data were obtained from glyvicinity for amino acids with any atom within 4.0  of any atom of a 
 carbohydrate moiety . in order to deal with any potential mistakes 
 that structures deposited in the pdb may 
 contain , which is a problem inherent in any attempt at gaining chemical 
 information from a public structural biology repository , the carbohydrate residues 
 within all of the pdb entries listed by glyvicinity were validated 
 with the privateer software , according 
 to the following criteria : first , only monosaccharides showing the 
 strongly preferred minimal energy conformation ( c1 for d - sugars , c4 for l - sugars ) were considered ; and second , only models with a good 
 fit to bias - minimized electron density were selected . only pdb entries 
 deposited along with structure factors  i.e . , experimental data  were 
 considered . the selected agreement metric was the real - space correlation 
 coefficient ( rscc ) , with a minimum cutoff value of 0.8 . as the significance 
 of this indicator decreases with decaying resolution , only entries 
 with a reported resolution of 2.0  or better were included . 
 of these , the coordinates of the monosaccharide and amino - acid residues 
 identified were extracted from the parent pdb files , where possible , 
 with examples where the nearby amino acids were identical ( as in homooligomeric 
 crystals ) discounted . the data set for each examined monosaccharide 
 was obtained using the glyvicinity assignment of the monosaccharide , 
 with erroneous assignments removed . for each monosaccharide class , 
 structures in which it was found were culled using cd - hit at 95% pairwise protein sequence identity , in 
 order to maximize the data available for each carbohydrate type while 
 minimizing bias from identical protein structures and point mutations . the relative occurrence of each amino acid in the vicinity of all 
 of the investigated monosaccharides was compared to that in the uniprotkb / swiss - prot 
 data bank . propensity = ( proportion of an 
 amino acid in the data set)/(proportion of that amino acid in uniprotkb ) ; 
 error bars represent 95% confidence assuming a normal approximation 
 of a binomial distribution . amino acids interacting with the 
 -/-faces were defined 
 as those where the center of the side chain was within 6  of 
 the ring atoms or c6 of the carbohydrate . ch 
 interactions were identified using three parameters 
 adapted from those previously used in a study of proteins . if multiple c h bonds fell within these 
 parameters for a single aromatic ring , that with the smallest c - projection 
 distance was taken as the primary interacting c minimized 
 conformations were generated from density functional theory ( b3lyp/6 - 31+(d ) ) 
 calculations in the gas phase using gaussian09 . fock ( b3lyp/6 - 31(d ) ) 
 energy calculations of these conformations at isovalue 0.002and visualized 
 using gaussview 5 . for the nmr 
 experiments , indole , 5-substituted indoles , and deuterium 
 oxide were obtained from sigma - aldrich and tci . glycosides ( other 
 than methyl--d - mannopyranoside , synthesis outlined 
 in supporting information ) were obtained 
 from pfanstiem and sigma - aldrich . proton 
 nmr spectra were acquired in d2o on a bruker avance-500 
 500 mhz spectrometer with a dch cryoprobe . experiments used a spectral 
 window from 11 to 1 ppm , a 4 s acquisition time , a 2 s relaxation 
 delay , and 64 scans . nmr experiments with a relaxation delay of 15 
 s were run to verify indole concentration . the shift of the trimethyl 
 peak of dss was normalized to dss = 0 ppm . for the 
 data points shown , three series of experiments were conducted at the 
 same glycoside and indole concentrations : indole only , glycoside only , 
 and mixed samples . the chemical shifts were averaged over three replicates , 
 and the chemical - shift perturbations were reported as  
 = indole  indole - free . total examples in data set . total proximal amino acids across 
 data set , and composition of these . average number of amino acids associated 
 with each carbohydrate face , and composition of these . carbohydrate 
 interactions , 
 first we used glyvicinity to create a 
 structural database of monosaccharide residues  i.e . , free monosaccharides , 
 or separated constituents of larger oligosaccharides  together 
 with proximal amino acids from x - ray crystal structures from the pdb . strict validation criteria were set to avoid incorporating entries 
 with incorrect nomenclature , unlikely 
 conformations , or poorly fitted experimental data . for the elucidation of interactions discussed herein , we 
 used the data in its broadest form : we chose 7 of the biologically 
 relevant carbohydrates that occurred most frequently in the data set , 
 as both - and -anomers , namely : d - glucose ( d - glc ) , d - galactose ( d - gal ) , d - n - acetylglucosamine ( d - glcnac ) , d - n - acetylgalactosamine ( d - galnac ) , d - mannose 
 ( d - man ) , d - xylose ( d - xyl ) , and l - fucose ( l - fuc ) . we treated each residue as an isolated 
 unit , considering only the pyranose form , and ignoring any modifications 
 of the hydroxyl groups ( e.g. , o - methylation , o - phosphorylation , etc . ) . we recognize that substituents 
 on the carbohydrate frameworks may well affect interactions , but our 
 focus on unmodified saccharide residues was simply to maximize the 
 available data and to find general , or first - order , interactions between 
 carbohydrates and their protein hosts . the resulting data set encompassed 
 carbohydrate moieties that could be divided into two groups : covalently 
 bound glycans ( from glycoproteins ) , and ligands bound noncovalently 
 to proteins , table s1 . an initial scan of the database indicated 
 that for glycans there were fewer close - contacts between carbohydrate 
 residues and protein side chains in glycosylated proteins than there 
 were for the same monosaccharides from ligands in protein for the four cases with sufficient 
 examples to allow comparisons/-d - man , 
 -l - fuc , and -d - glcnac  the covalently 
 bound carbohydrates made on average approximately one - half to two - thirds 
 the number of contacts with protein side chains , and less than one - fifth 
 of the ch interactions , than observed for the corresponding 
 noncovalent complexes . these differences are perhaps not surprising , 
 as the covalent linkage in glycoproteins does not require effective 
 noncovalent interactions to bind the carbohydrate to the protein . an interesting additional possibility , however , is that such interactions 
 may be less likely to occur in glycoproteins , where the glycan can 
 participate in intermolecular proteincarbohydrate interactions 
 as an alternative . thus , the saccharide s most - effective binding 
 face is not occluded through an intramolecular interaction , but rather 
 left free to engage in an intermolecular interaction . without binding 
 partners present in the x - ray crystal structures , whether such trade - offs 
 occur can not be seen . whatever the reasons for the lower density of 
 protein  carbohydrate interactions in the glycans , we focused 
 our subsequent analyses on noncovalent protein carbohydrate 
 complexes , table 1 , 
 as we were interested in the interactions of carbohydrate ligands 
 for this study . the amino acids proximal to carbohydrates were normalized 
 to their occurrence in all protein sequences , figure 1 . independent of the method of normalization 
 employed ( figure s1 ) , three trends emerged . first , we observed only a small preference for polar , hydrogen - bonding 
 residues within these binding sites ; although of these residues , aspartic 
 acid ( asp ) and asparagine ( asn ) were particularly favored , occurring 
 approximately twice as often as expected by chance . second , and without 
 exception , aliphatic residues were disfavored in carbohydrate - binding 
 pockets . this exclusion would not be expected if the hydrophobic effect 
 alone played a major role in carbohydrate binding . third , and most 
 conspicuously , three of the four aromatic residues contacted carbohydrates 
 more frequently than expected by chance , in the order tryptophan ( trp ) 
  tyrosine ( tyr ) > histidine ( his ) . these last two observations 
 highlight that carbohydrate  aromatic interactions are a key 
 defining characteristic of carbohydrate - binding sites , whereas , hydrophobic 
 interactions per se are not . they also reveal that not all aromatic 
 residues are equivalent  some are more likely than others to 
 interact with carbohydrates . we examined the aromatic residues that we identified 
 in detail , postulating that the juxtapositions of carbohydrate and 
 aromatic residues should illuminate the forces that drive protein we illustrate our observations and 
 arguments with comparisons between two well - represented isomers , -d - glc and -d - gal , that differ in stereochemistry 
 at only the 4-hydroxyl group , figure 2a , d . the general and discriminating features emerging 
 from this comparison are emblematic of those that we observed more 
 broadly for carbohydrate we compared amino - acid distributions 
 around -d - glc and -d - gal by first 
 focusing on the two distinct surfaces of carbohydrate rings , the - 
 and -face , figure 2a , d . h bonds that differ 
 in stereochemistry and stereoelectronics between monosaccharides configurations . 
 with its completely equatorial arrangement of hydroxyl and alkoxyl 
 groups , -d - glc has approximate symmetry , with a polar 
 perimeter in the plane of the saccharide ring bisecting the - 
 and -faces consisting of c h 
 bond arrangement , we found similar numbers of aliphatic and aromatic 
 contacts on the -face , and a slight ( 2.7-fold ) preference for 
 aromatic over aliphatic residues on the -face , figure 2b , video s1 and table 1 . we quantified the proportions of side chains nearest each carbon 
 of the carbohydrate to determine how different c our observations largely tracked the direction 
 of the c  h bond , with a higher preference for aromatics and 
 aliphatics on the face toward which the c  h bond was oriented . for example , contacts to both aromatic and aliphatic side chains on 
 the -face were made by c(2)h and c(4)h ; those 
 made on the -face were largely effected by c(1)h , c(3)h , 
 and c(5)h , whereas c6 failed to exhibit a facial preference , 
 presumably because of rotation around the c5c6 bond . amino acids 
 proximal to carbohydrates in x - ray crystal structures 
 of protein  carbohydrate complexes . propensities of amino acids 
 ( in order of increasing hydrophobicity ) in carbohydrate - binding sites from the data set compared to the 
 distribution of amino acids across all proteins in uniprot . alternative methods for normalization are given 
 in figure s1 ; however , the overall trends 
 shown here are preserved . color code : white , hydrogen - bonding side 
 chains ; gray , aliphatic hydrophobic side chains , including gly , pro , 
 cys and met ; beige , aromatic side chains . in contrast , -d - gal exhibited marked differences 
 in amino - acid environment between the - and -faces , table 1 , figure 2d  f , video s2 . these findings underscore the importance of the 
 carbohydrate stereochemistry , as the change in configuration at the 
 c4 position has a major effect on interaction with aliphatic and aromatic 
 amino acids . in detail , aliphatic residues were largely excluded from 
 the -face of -d - gal , but aromatic side chains 
 were prevalent , with a 14-fold preference for aromatic moieties . this 
 preference was especially strong at the c(4)h and c(5)h 
 positions , figure 2f , and was much starker than that observed for -d - glc c  h protons , indicating more - favorable interactions with 
 aromatics . analogous variations in c  h bond interactions 
 were seen 
 for other monosaccharides , figure s2 . for 
 example , for -d - glc the only axial hydroxyl is on 
 the -face , the reverse case to -d - gal . correspondingly , 
 opposite to -d - gal , we found a high preference for 
 c  h bonds to interact with aromatic residues on the -face 
 of -d - glc , but little discrimination for those on 
 the -face , figure s2a . ( a  c ) -d - glc , and ( d  f ) -d - gal . ( a , d ) - and -faces and ring c  h 
 bonds . ( b , e ) centers , represented as spheres , of aromatic and aliphatic 
 side chains interacting with the faces of the carbohydrates ( i.e. , 
 within 6  of any carbohydrate carbon or the ring oxygen ) . ( c , 
 f ) proportions of aromatic and aliphatic side chains interacting with 
 the - and -faces reported to the nearest carbon atom 
 of the pyranose ring . h bonds that seem chemically similar , such 
 as the 
 c(4)h bonds of -d - glc and -d - gal , have different preferences for interaction with aromatic moieties . furthermore , preference for aromatics is at the expense of aliphatic 
 amino acids , further discounting the hydrophobic effect as an explanation . aromatic 
 interactions by investigating the electrostatic potentials of the 
 aromatic moieties and carbohydrate c  h bonds . unlike aliphatic residues , 
 aromatic amino acids present electronegative 
 -electron systems above and below the planes of the aromatic 
 rings that can interact with carbohydrate c  h bonds through 
 ch interactions . we posited 
 that if electrostatic contributions are important for ch 
 interactions in protein carbohydrate complexes , differences 
 in the electronics of the aromatic systems and carbohydrate c we identified 
 ch interactions in the data set using a three - parameter 
 operational definition for the interaction ( figure 3a ) , and 
 then we probed for any correlations between the electronics of aromatic 
 and carbohydrate rings , calculated and visualized as electrostatic 
 surface potentials ( esps ) , at the sites of the interactions . we found that across our database the four aromatic side chains engaged 
 in ch interactions with carbohydrate c h bonds 
 to different extents , with the order trp > tyr > phenylalanine 
 ( phe ) 
 > this 
 ranking reflects the esps of these side chains ( figures 3c and s4a  i ) and implies that electron - rich aromatic systems are the most likely 
 to engage in ch interactions . the aforementioned 
 ranking could stem solely from the relative 
 surface areas of the aromatic side chains . when normalized for surface 
 area of the -systems , however , the most electron - rich trp remained 
 the most common acceptor of ch interactions figure s5 . the aromatic systems of tyr and phe both present a similar 
 surface area , comprising 6-carbon - membered rings . indeed , a study 
 of such interactions between amino acids within protein crystal structures 
 found phe and tyr were equally likely to participate as ch 
 acceptors , possibly highlighting differences 
 for intra- and intermolecular systems . in terms of electronics the 
 two systems are not equivalent . participation of the tyr hydroxyl 
 in hydrogen bonding as an h - bond donor  which is the case for 
 almost all examples of tyr in proteins increases the electron - density of the -system of tyr , figure s4 . as shown by the esps , figures 3c and s4c f , this increases the electronegativity of the 
 -system , hence making it a preferred acceptor over phe . trp 
 is almost always involved as an h - bond donor in proteins , which increases the electronegativity of the 
 -system beyond h - bonded tyr , figure s4a , b . interpretation of the data for the side chain of his is complicated 
 by the different hydrogen - bonded and protonation states that it can 
 take ; however , its involvement in ch interactions in 
 protein  carbohydrate complexes , figure 3b , and proteins in general , is relatively small . ( a ) parameters used to identify ch 
 interactions : ch angle 
 (  ,  40 ) , ch distance ( c - x ,  
 4.5  ) , c - projection distance ( cp  x ,  
 1.6  for his and trpa ;  2.0  for phe , trpb , tyr ) . ( b ) raw - count distribution of aromatic side chains identified making 
 ch interactions with carbohydrates . for trp , ch 
 interactions were identified for cases where either the five- or six - membered 
 ring interacts with a ch proton , trpa and trpb , respectively , and 
 where the two rings both interact with separate ch protons , trpa+b . ( c ) structure of proteinogenic aromatic amino acids , with corresponding 
 electrostatic surface potentials for the -systems ( highlighted 
 in beige ) of the side - chain moieties : indole ( trp ) ; phenol ( tyr ) ; 
 benzene ( phe ) ; imidazole ( his ) . for indole and phenol , the forms as 
 hydrogen - bond donors ( h - bonded to water ) are shown , as these are predominant 
 in protein x - ray crystal structures . to 
 show the differences in the -systems , the scale is shown from 
 130 kj mol ( electropositive , blue ) through 
 neutral ( green ) to  130 kj mol ( electronegative , red ) . it is striking that the ranking of aromatic amino acids involved 
 in ch interactions closely aligns with that observed 
 for cation- interactions in similar ligand binding systems . for many cation- interactions , such as 
 those of the tetramethylammonium cation , the interaction of the positive 
 charge with electron - rich aromatic rings is mediated by c h 
 protons , and this could be argued to 
 be analogous to a ch interaction involving extremely 
 polarized c  h bonds . next , we investigated whether involvement in ch 
 interactions also depended on the electronics of the carbohydrate 
 c  h bonds . such preference could contribute to carbohydrate 
 discrimination : the positivity of the carbohydrate c  h protons 
 results from the overall hydroxyl stereochemistry . therefore , to compare 
 the c  h protons , we examined the esps of the different monosaccharides 
 in more detail . aromatic 
 interactions are already known to play key roles in its binding ; and indeed , of all the well - represented monosaccharides , 
 our analysis revealed that it made the highest proportions of ch 
 interactions , table 1 . while steric hindrance can impact the ability of some c  h 
 bonds ( e.g. , c(2)h ) to participate in ch interactions , 
 the data suggested electronic effects are critical . the configuration 
 of the hydroxyl groups of -d - gal give a cluster of 
 c  h bonds on its -face , formed by c(1)h , c(3)h , 
 and c(5)h and extending to the edge where c(4)h and 
 one of the c(6)h atoms are located , figure 4b . while often described as a nonpolar 
 patch  , the esp indicates that it is in fact partially positive , 
 and this  positive patch  corresponds to the area where 
 interacting side chains are almost exclusively aromatic , figure 2e , f . one way to rationalize 
 this particularly electropositive patch is through stereoelectronic 
 effects leading to more positive c  h protons : the axial c4-hydroxyl 
 withdraws electron density from c3 and c5 protons via overlap of the 
 c  h  orbital with the  * orbital of the c(4)o 
 bond , and the c4 proton is rendered electron - poor through overlap 
 with  * orbital of the ring c  o bond . superposition 
 of the subset of aromatic side chains engaged in 
 ch interactions revealed them located predominantly 
 over the most electropositive c very few examples interacted with the 
 c(2)h of the -face , for which the electrostatic potential 
 is more neutral . that the more - positive protons of the carbohydrate 
 interact more frequently with the electron - rich aromatic systems is 
 consistent with a contribution from electrostatics to ch 
 interactions . to test the importance of electronics more generally , 
 we compared 
 the esps of further carbohydrates and assessed their engagement in 
 ch interactions , figures 5 , s6 and s7 . in 
 all cases , our findings support a role for an electrostatic contribution 
 to the ch interactions . as the electronics of the carbohydrate 
 c h bonds are determined by the identity of the monosaccharide 
 and the anomer , this leads to distinct modes of interaction for the 
 different classes . for example , -d - gal and -d - glc more often than not engaged in ch interactions 
 with proximal aromatic residues ; however , such contacts were less 
 common in binding sites of -d - man , -l - fuc , -d - xyl , and - and -d - glcnac , which do not present such electropositive c ( a ) orthogonal views 
 of a minimized conformation of -d - gal , representative 
 of the majority of those found in the database , which has the -angle 
 favored by gal in solution and in protein crystal structures , in stick - model representation with c  h 
 protons numbered systematically . ( b ) esp calculated for the minimized 
 conformation . to show the differences in the c h bonds , the 
 scale is shown from 260 kj mol ( electropositive , 
 blue ) through neutral ( green ) to  260 kj mol ( electronegative , red ) . this is double that used for the aromatic 
 systems ; i.e. , similar changes in color here signify bigger differences 
 than in figure 3c . ( c ) juxtaposed aromatic moieties of amino acids engaged in ch 
 interactions with -d - gal . the -faces of -d - glc and -d - gal isomers are sterically similar , figure 5a , b , and yet the propensity for the two carbohydrates 
 to engage in ch interactions on this face differed . this is because the -face c  h protons are comparatively 
 more electropositive for -d - gal , which should promote 
 ch interactions , particularly those involving the c4 
 and c5 protons , figure 5a . 97% of ch interactions occurred on the -face 
 for -d - gal , at an average of almost one interaction 
 per example , table 1 . the corresponding -face protons of -d - glc 
 are less electropositive , and , as a result , ch interactions 
 were less frequent , figure 5b . 68% of interactions occurred on the -face for -d - glc , just over 0.5 per example on average . examination 
 of other , albeit less - well represented , monosaccharides 
 in our database provided further support for electronic effects , figures s6 and s7 . for example , for both -d - gal and -d - glc the axial hydroxyl on the -face 
 reduces the electropositivity , and correspondingly , there are ch 
 interactions , of the -face c  h bonds compared to the 
 -anomers , figures s6a , c and s7a , c . for -d - glc the most positive c h bonds are 
 on the -face , and this is where most ch interactions 
 occurred , figure 5c . disruption or reduction of the electropositive patches led to lesser 
 involvement in ch interactions . for -d - man , the 1,2-diaxial arrangement of hydroxyl groups prevent there 
 being any very electropositive c  h protons , figures s6e and s7e . the ch interactions 
 of -l - fuc also 
 suggested a contribution of electrostatics over hydrophobic or simple 
 steric effects particularly well : the lack of oxygen at c6 relative 
 to -d - gal reduces the electropositivity of the c h 
 protons at c5 and c6 , and correspondingly fewer ch 
 interactions , despite fuc being the more hydrophobic overall , figures s6 m and s7 m . finally , and as an experimental test , we probed 
 how our two exemplar carbohydrate residues , -d - glc 
 and -d - gal , interacted with aromatic residues in aqueous 
 solution . we used h nmr spectroscopy to follow the association 
 of indole ( as a trp surrogate ) and the two -methyl - glycosides . 
 in both cases , there were small but measurable and reproducible upfield 
 shifts ( negative  ) indicative of ch interactions of some , but not all , c  h protons of 
 the carbohydrates , figures 6a , s8 , and s9 . moreover , the magnitudes 
 of the changes differed between protons , with the nmr data , figures 6a and s9 , in good agreement with the database - derived 
 propensities , figure 5 . as predicted , carbohydrate  aromatic interactions were stronger 
 for -methyl - d - gal than for -methyl - d - glc . for the former , larger chemical - shift changes were observed 
 for the c1 , c3 , c4 , and c5 protons , i.e. , all on the electropositive 
 -face of the monosaccharide . the interactions with -methyl - d - glc were weaker , consistent with a less - electropositive -face 
 and our database analysis , figures 6a and s9 . indole gave stronger 
 ch interactions than previously reported for phenol 
 or benzene , in accord with the observed 
 preference for trp in carbohydrate - binding sites , table s3 . our findings are in accord with those of others 
 on model peptides , and between methyl 
 glycosides with the free amino acids l - phe , l - trp , 
 and l - tyr . again , these data 
 suggest that the favorable ch interactions make critical 
 contributions to the binding of some but not all saccharides . our analyses of the esps suggested that other saccharides , less - well 
 represented in our bioinformatics study , also present clusters of 
 electropositive c one such carbohydrate epitope is -d - man . 
 because of the axial c(2)oh , the -face c  h bonds 
 of -d - man ( at c1 , c2 , c3 and c5 ) form an electropositive 
 patch analogous to that of -d - gal , figure s6f therefore , we postulated that -d - man should engage in ch interactions at these positions . this hypothesis was supported by the relatively small number of examples 
 in our structural database , table 1 . by h nmr we detected similar ch 
 interaction strengths as those observed for -methyl - d - gal . as predicted , the indole interacted with the most - electropositive 
 c  h protons on the -face of -d - man , figures 6a and s9 . ( a ) -d - gal , ( b ) 
 -d - glc , and ( c ) -d - glc . column 1 : 
 stick models for representative minimized conformations viewed from 
 the -faces with c  h protons numbered . column 2 : normalized 
 calculated esps for the same orientation of the minimized conformation . the scale is shown from 260 kj mol ( electropositive , 
 blue ) through neutral ( green ) to  260 kj mol ( electronegative , red ) ; as with figure 4b this is double that used for the aromatic 
 systems in figure 3c . column 3 : the distributions of aromatic side chains that form 
 ch interactions with the monosaccharides . column 4 : 
 average frequency of involvement of the monosaccharide c  h 
 protons in the ch interactions . for complete analyses 
 for all monosaccharides see figures s6 and s7 . to examine further electronic 
 effects in the associations in solution , 
 we carried out a linear free energy ( hammett ) analysis of the binding 
 of methyl--d - gal to different 5-substituted indoles , figures s10 and s4 . we monitored changes in chemical 
 shift for the most perturbed gal ring proton , c(5)h , figure 6b . electron - rich 
 indoles gave larger changes in chemical shift than did indole itself , 
 indicating that the former engaged in stronger ch interactions . in contrast , electron - poor indoles afforded weaker interactions , and 
 the strongly electron - withdrawing nitro - substituent appeared to abolish 
 the interactions entirely . the linear trend observed , figure 6b , indicates that electronic 
 effects are critical in ch interactions . in summary , we provide a quantitative assessment 
 of the interactions made between 
 protein side 
 chains and the pyranose forms of the most - common monosaccharides found 
 across all high - resolution structures of protein  carbohydrate 
 complexes in the protein data bank . we have quantified biases in the 
 amino - acid occurrence in the immediate vicinities of the carbohydrates , 
 with a preponderance of aromatic residues , and particularly the electron - rich 
 side chain of tryptophan , above and/or below the plane of the carbohydrate 
 rings . thus , it is not simply the case that the faces 
 of the carbohydrate are sequestered through the hydrophobic effect . our data indicate that two effects are at play . as a first - order effect , 
 the electronegative faces of the aromatic rings engage in favorable 
 electrostatic interactions with certain electropositive faces of the 
 carbohydrates . in addition , a more - specific and more - intimate second - order 
 effect operates . h 
 bonds of the carbohydrate engaging in ch interactions 
 with a contacting aromatic ring . this model is supported by calculation 
 of the electrostatic surface potentials of both the carbohydrate and 
 arene rings , examination of the proximity of individual carbohydrate 
 carbon atoms to the aromatic groups , and the linear free energy relationship 
 analysis . moreover , because the electrostatic surfaces , and , importantly , 
 the electropositive characters of c h bonds differ between 
 carbohydrate isomers , the aromatic side chains engage with different 
 regions of the carbohydrate . this not only provides a mechanism contributing 
 to the binding of carbohydrates by proteins , but also for discriminating 
 between one monosaccharide and other closely similar structures within 
 their binding sites . the circle color and size is 
 scaled to represent the chemical - shift change relative to indole - free 
 solutions (  = indole  indole - free ) . from left to right : -d - gal , -d - glc , and -d - man . ( b )  
 shift for h5 and methyl c  h protons of methyl--d - gal versus the hammett p parameter of the 5-substituent 
 in a series of substituted indoles . to allow for solubility limitations , all perturbations were normalized to 7.5 mm indole using the linear 
 dependence of chemical - shift perturbation on indole concentration , figure s9 . linear fits of the data are shown 
 for h5 ( gradient = 5.7 , r = 0.86 ) and 
 me ( gradient = 2.1 , r = 0.63 ) . these bioinformatics and experimental 
 findings provide a strong 
 construct for understanding the fundamental forces underpinning protein carbohydrate 
 interactions , and they have implications for studies of their molecular 
 recognition . for instance , by increasing the electropositivity of 
 c  h bonds , carbohydrate binding should be facilitated via improved 
 carbohydrate  aromatic interactions . in this way , carbohydrates 
 with electron - withdrawing o - acylated or o - sulfated groups could form stronger ch interactions . similarly , hydrogen bonding or calcium - ion coordination to key carbohydrate 
 hydroxyl groups could increase the strength of ch interactions . given the vital role that carbohydrate  protein interactions 
 play in biology , one strategy for designing glycomimetic drugs would 
 be to exploit specific ch interactions , or the general 
 presence of electron - rich aromatic rings to complement electropositive 
 faces of carbohydrates in binding sites . while the importance of ch 
 interactions in carbohydrate - based environments is apparent from our 
 studies , this class of interaction plays roles within wider ligand 
 binding , the structure of macromolecules and proteins , and in the 
 mechanisms of chemical reactions . therefore , 
 appreciation of the impact of stereoelectronic effects on these and 
 similar noncovalent interactions has potential for application within 
 many contexts .
protein  carbohydrate interactions 
 play pivotal roles in 
 health and disease . 
 however , defining and manipulating these interactions 
 has been hindered by an incomplete understanding of the underlying 
 fundamental forces . to elucidate common and discriminating features 
 in carbohydrate recognition , 
 we have analyzed quantitatively x - ray 
 crystal structures of proteins with noncovalently bound carbohydrates . 
 within the carbohydrate - binding pockets , 
 aliphatic hydrophobic residues 
 are disfavored , whereas aromatic side chains are enriched . 
 the greatest 
 preference is for tryptophan with an increased prevalence of 9-fold . 
 variations in the spatial orientation of amino acids around different 
 monosaccharides indicate specific carbohydrate c 
 h bonds interact 
 preferentially with aromatic residues . 
 these preferences are consistent 
 with the electronic properties of both the carbohydrate c  h bonds and the aromatic residues . 
 those 
 carbohydrates that present patches of electropositive saccharide c  h 
 bonds engage more often in ch interactions involving 
 electron - rich aromatic partners . 
 these electronic effects are also 
 manifested when carbohydrate 
 aromatic interactions are monitored 
 in solution : nmr analysis indicates that indole favorably binds to 
 electron - poor c 
 h bonds of model carbohydrates , and a clear 
 linear free energy relationships with substituted indoles supports 
 the importance of complementary electronic effects in driving protein  carbohydrate 
 interactions . 
 together , our data indicate that electrostatic and electronic 
 complementarity between carbohydrates and aromatic residues play key 
 roles in driving protein 
 carbohydrate complexation . 
 moreover , 
 these weak noncovalent interactions influence which saccharide residues 
 bind to proteins , and how they are positioned within carbohydrate - binding 
 sites .