Some nuclei properties important for NMR detection.
1.1. NMR spectroscopy: Approaching glycobiology
Generally speaking, nuclear magnetic resonance (NMR) spectroscopy seems the most powerful technique in current use for structural analysis of biomolecules. Four Nobel prizes have been awarded so far due to discoveries related to NMR: 1952 (Physics) to Felix Bloch and Edward Mills Purcell for explanations of the physical properties of nuclei under magnetization; 1991 (Chemistry) to Richard Ernest for the development of the principles for the multidimensionality in NMR spectroscopy; 2002 (Chemistry) to Kurt Wüthrich for applying NMR in structural determination of biomolecules, mainly proteins; and 2003 (Medicine) for Paul Lauterbur and Peter Mansfield to the discoveries concerning the use of magnetic resonance imaging in medical diagnostics. The significant boom of NMR spectroscopy in structural biology however dates from the beginning of 80´s, mainly due to the implementation of two-dimensional techniques associated with advances in instrumentation and
Particularly, we could generalize that in the following two decades, proteins and nucleic acids were the primary biomolecule types in NMR studies. This was somewhat related to the usual 3D structures these molecules may present in solution. This consequently facilitates the detection of valuable spatial contacts between residues that are displaced away in a polymeric chain. The achievements by NMR in studies of proteins and nucleic acids were also significant to push the genome and proteome projects. Although, carbohydrates were also analyzed by NMR at this period, the association between glycobiology and NMR was somewhat neglected. For sure, this has happened because of the general idea of high structural complexity of carbohydrates combined with their high-order dynamic properties (high molecular flexibility). The conception of an unclear or absent three-dimensional states for carbohydrate molecules was one of the greatest reasons to this limited attention.
However, persistent research groups worldwide have been proving otherwise and showed to the community interesting NMR results concerning glycans. These results have helped to unravel the biological roles of glycans. Above all, since that time, glycobiology turned to be a fertile field for NMR spectroscopy. Due to the current glycomics´ boom, the high number of vital biological roles triggered by carbohydrate molecules, and the advances in NMR methods, NMR and glycobiology are now a very promising combination. Hence, in this chapter, we want to illustrate this new trend. For this, we cover the basis of the major solution NMR methods in glycobiology, taking examples from our experiments or from published works. We offer to the readership the adequate conclusions of these NMR data and moreover, what they represent for explaining the physicochemical mechanisms of the glycans addressed.
2. The essential high-throughput NMR methods exploited in glycobiology
NMR spectroscopy has a special position compared to the other analytical techniques once it provides a variety of experiment types to be performed as many as structural features to investigate in a biologically relevant macromolecule. Moreover, the NMR studies can be performed in solution which can mimic the physiological conditions. The major disadvantages in application of NMR spectroscopy are that of sensitivity and limitation in high-molecular mass systems. In order to overcome respectively these two obstacles, methods of
Among many NMR techniques to be applied in glycobiology, we shall explain the most basic and utilized ones, such as 1) information from different NMR-active isotopes (1H, 13C, 15N); 2) cross-peak assignments through multi-dimensional NMR spectra for tabulating chemical shift values or structural characterization purposes; 3) dynamic studies by measurements of longitudinal or spin-lattice (T1) and transversal or spin-spin (T2) relaxation rates (R1 and R2, respectively, in ms to s), and of line-widths or line broadening (in Hz) in certain NMR timescale; 4) chemical shift changes (in ppm) that may reflect conformational changes in carbohydrates due to temperature variations, or in carbohydrate-binding proteins in the presence of ligants where localized chemical shift perturbation is induced by presence of intermolecular contacts; 5) measurements of constants of either scalar (
2.1. The NMR-active isotopes mostly used in carbohydrate studies
The three isotopes with magnetically active nuclei spin-½ mostly studied in glycobiology NMR are 1H, 13C, and 15N. Each one has its own magnetic relative susceptibility (Table 1), its own precessional frequency (Larmor frequencies at 800 MHz for 1H, for example, in Table 1), relaxation properties, and principally differential atomic positions within a molecule. These differenced localizations ultimately provide useful information concerning structural features, in the atomic perspective, and dynamic properties, of specific regions or sites within a molecule. In dynamic studies, localized motions as well as global motions of the analyzed molecules can also be evaluated, depending on the atom or group of atoms that have to be examined. It is worth mentioning that the relative NMR receptivity (dependent on the isotopomeric abundance, relaxation and magnetogyric ratio) of these three biomolecularly abundant NMR-active isotopes is quite different, which leads to different NMR sensitivity. While 1H has its value set as 1.0, 13C and 15N have their values as 1.76x10-4, and 3.85x10-6 (Table 1).
|Nuclide||Spin||Natural abundance||Gyromagnetic ratio γ [107 rad T-1 s-1]||NMR Frequency (at 18.8 Tesla)||Relative receptivity|
|Proton (1H)||½||99.985||26.7522||799.734 (1)||1.00|
|Carbon-13 (13C)||½||1.108||6.7283||201.133 (1/3.976)||6.73x10-7|
|Nitrogen-14 (14N)||1||99.63||1.9338||57.820 (1/13.831)||1.00x10-3|
|Nitrogen-15 (15N)||½||0.37||-2.7126||81.093 (1/9.861)||3.85x10-6|
In principle, one would say it would be one of the greatest luck for NMR spectroscopists, the existence of biomolecules, including carbohydrates, rich in sensitive and active isotopes for NMR studies (Table 1). This is true if we consider the essential magnetic properties and abundance of hydrogen atoms in such molecules. Coincidently or not, in proteins, nucleic acids, lipids, and not differently in carbohydrates, hydrogen is not the most abundant atom, but it also fundamentally participates directly (by physical contacts) in many of the biological reactions, either through hydrogen-bond networks in protonated states during binding events, or even indirectly because of its absence in deprotonated states during pH-changes occurred during physiological reactions. Therefore, one would guess that 1H-atoms would be the most used isotope in NMR analysis, and this is exactly what happens not only for glycomics but also for lipidomics, proteomics, and genomics. The changes and profiles of 1H-signal distribution are informative in terms of molecular states, reactions, and dynamics, as exemplified by the simple mutarotation changes of the anomeric hydrogens of reducing terminal sugars in solution as a function of time (Figure 1). For example, only a simple structural change at the C2 of the glucose (Glc)-derived monosaccharides is enough to cause kinetic alterations of the 1H-anomerics equilibrium in solution. The intensities of the 1H-peaks properly indicate the proportion of these protons within a molecule, and therefore they are extremely useful to estimate the atoms and the percentage of each enantiomeric form (percentages in Figure 1). Actually, just after mass spectrometry (MS) techniques, measurements based on integrals of 1H-signals seem to be the most reliable quantitative procedure to determine the number of atoms within a molecule, as well as their conformational states.
The profile of 1H-signals in 1D 1H-NMR spectra offers a general fingerprint of the biomolecule in a solution such as the degree of pureness crucial for carbohydrates with biomedical purposes. However, due to the large density of protons in biomolecules, including carbohydrates, sometimes the 1H-NMR profile is overwhelming because of the many condensed, overlapped and thus unresolved signals. Therefore, the use of other nuclei such as 13C and 15N, either coupled or uncoupled to 1H, becomes valuable and complementary in the analyses, resulting important conclusions with respect the physicochemical properties of carbohydrates. For example, the intensity of 13C-signals of anomerics can also be diagnostic for the changing rates of the α- and β-states during mutarotation in solution, as demonstrated below. The directly observed 13C-signals are frequently much sharper than 1H-signals due to its differential relaxation property. The thinner 13C-signals in 1D NMR profile result usually more well-resolved peaks, and through the unidimensional scale, less superimpositions should occur. This is exactly what happens in structural analyses of algal sulfated polysaccharides with high degrees of structural heterogeneities [12-14]. 1H-coupled 13C-signals are very diagnostic of the methine (CH), methylene (CH2) and methyl (CH3) groups highly abundant in carbohydrate molecules, and thus quite useful in NMR structural glycobiology. In case of carbohydrates or glycosylated molecules that contain amino sugars, like the glycosaminoglycans (GAGs) that bear hexosamines with 15N-natural abundant amino groups, the 15N-related NMR signals although few in number are well-resolved and still quite useful for structural diagnosis . Hence, hetero-atoms are very useful in solution NMR structural glycobiology through multi-dimensional heteronuclear experiments, or in direct observe experiments, as discussed next to describe the assignments of glucose and the novel method for characterizing GAG molecules through 15N-atoms.
2.2. Peak assignments through multi-dimensional NMR spectra of glycans
Although 1H-NMR spectra are quite informative in terms of the general view of the molecule and diagnostic for the presence of contamination, fully protonated molecules with high-molecular weights, and with a certain degree of heterogeneity, result in very complex 1H-NMR spectra. This can be seen by the presence of many unresolved peaks from polysaccharides with no clear pattern of structural regularity. In general, in glycan analysis, except the 1H-signals belonging to the anomerics that resonate at the most downfield region of the spectra (usually located somewhere between 4.5 and 6.0 ppm), all other ring protons from the most monosaccharide types resonate very squeezedly between 3.0 and 4.5 ppm. This can be seen through the 1D 1H-NMR spectrum in solution of the simple and most common monosaccharide, the glucopyranose (Glc
Observing either the 1D 1H-NMR spectrum (Figure 2B), or the 2D 1H-based NMR spectra (Figure 3A, and 3B), it is clear that the anomeric 1H-signals at the downfield region of the suppressed water (HOD) peak at ~4.50 ppm are well-resolved, and thus they may serve as good starting-points to trace connectivities using 2D-NMR spectra. Usually, α-1H-anomeric signals resonate more downfield than β-ones in the majority of the carbohydrate units. In the case of glucose, the α-1H signals resonate exactly at 5.32 ppm, while the β-1H signals resonates exactly at 4.74 ppm (Table 2).
Since COSY experiments create 1H-1H cross-peaks within 3JH(n)-H(n+1) (scalar couplings of protons of three-bond distances) like those observed for 3
The downside in collecting spectra containing heteronuclear-filters (either 13C-, or 15N-) even with acquisition through 1H, such as in the HSQC experiment (Figure 3C) is that it is more time-consuming than those based exclusively on 1H-atoms. This is because of the sensitivity and abundance of the heteronuclear isotopes, which are much lower than those of proton (see NMR receptivity in Table 1). This can be proved by different signal-to-noise ratio clearly seen by the baseline widths comparatively observed in the 1D 1H- and 13C-NMR spectra (Figure 2B vs 2C). In 13C-directely observed NMR spectra, the peaks are usually thinner compared to 1H-signals (Figure 2B vs 2C). This is, in turn, due to different relaxation properties of the 13C-nucleus. The longitudinal or T1 relaxation of carbons is much faster than protons, which result a considerably sharper peak in 1D NMR (Figure 2B vs 2C).
Due to the lower relative receptivity of heteronucleus, sometimes isotopic labeling techniques are necessary to develop 2D NMR analysis for certain carbohydrates. This is crucial, especially for those cases where the amount of material is a limitation, such as glycans isolated from cell cultures. Recently, we developed an
Note that through 15N-HSQC spectroscopy, all GAG standards show just the 1H-15N cross-peak related to their differential hexosaminyl units, in a very simple way that allows rapid structural assignments. Although the NMR peaks are few, these resonances are still quite useful for structural determination in glycosaminoglycanomics. All signals from different standards are characteristic, and resonate with distinct 1H- or 15N-chemical shifts (Figure 4) . Structural features such as the hexosaminyl type (galactosamines at upfield 1H- and 15N-chemical shifts, Figures 4A-D, as opposed to glucosamines at a more downfield 1H- and 15N-chemical shifts, Figure 4E), sulfation pattern (4-sulfation at the upfield 15N-chemical shifts, Figures 4A, B and D, as opposed to more downfield 15N-chemical shifts of 4,6-di-sulfated units, Figure 4C, and further more downfield 15N-chemical shift of 6-sulfated units, Figure 4A, and B), and adjacent uronic acid type (glucuronic acid at upfield 1H-chemical shifts, Figures 4A-C, as opposed to iduronic acid at the more downfield 1H-chemical shifts, Figure 4D) can be easily determined through this method . These studies concerning the use of 15N-NMR for structural characterization in GAG molecules turned out to be quite valuable also in predicting the anomericities in GAG-derived oligosaccharides as well as the sulfation patterns. For more details read reference 10 about this topic.
One downside of the 15N-NMR application on structural studies of GAGs is the very fast exchange rates of protons from sulfamate groups (NHSO3-) that are quite abundant in glucosamines of certain GAG types, such as heparan sulfates and heparins. Although the 1H-15N cross-peaks from 15N-HSQC spectra of residual N-acetylated glucosamines (GlcNAc) can be fairly used to quantify the amounts and types of uronic acid units, to which GlcNAc are linked (Figure 5), the remaining amounts of uronic acids linked to N-sulfated glucosamines (GlcNS) can be missed under normal conditions. Alternative ways to force the protonated states in sulfamate groups is the use of controlled samples at narrow pH range (7.0-8.0) , or to slow down the fast 1H-amide exchange rates recording experiments at very low-temperatures, as detailed next with preliminary results using commercially available sodiated GlcNS as a molecular model to mimic the composing N-sulfated amino sugars in heparins and heparan sulfates.
Very low temperatures such as 3oC (20% acetone is added to avoid freezing) can slow down considerably the solvent exchange of the labile protons. All protons, inclusively the exchangeable ones from sulfamate or hydroxyl groups of GlcNS, become thus detectable by 1H-based NMR spectroscopy (Figure 6A). This can be exemplified with the 1D spectrum of GlcNS at Figure 6A. The 1H-peaks can be assigned through spin-systems from TOCSY spectrum using this same hydrated low-temperature condition (Figure 7A), like the same way undertaken and explained for Glc (Figure 2). The α- and β-anomers with 1H-chemical shifts respectively at ~ 5.42 and ~ 4.68 ppm (Figures 1 and 6A) serve as starting point for tracing connectivities by cross-peaks in TOCSY spectrum also recorded in water-rich solution (Figure 7A). They have 3
2.3. Examining flexibility and dynamic properties of glycans by relaxation rates measurements
In radio-frequency pulsed NMR experiments, the magnetization aligned with the static magnetic field (B0) is titled away from the longitudinal
In a recent study about structural dynamics of the saccharidic portion of immunoglobulin G (IgG) using NMR spin-relaxation , the authors have proved that both glycan branches at the Fc fragment (Figure 8) are accessible and dynamic in solution. This motion, and glycosylated rates (Figure 8) are responsible to participate during the cellular responses of the adaptive immune systems, mainly modulating the health balance between hyposensitivity to foreign particles versus hypersensitivity to auto-antigens. Again, the spin labeling technique as discussed in section 2.2 was crucial to advance this work. In this reference, the N-glycan at Asparagine-297 was enzymatically remodeled by sialytransferases and glycosidases to build up branches specifically labeled with 13C-isotope in galactosyl units (Figure 8). This initial procedure of spin labeling indicated already an apparent accessibility of these branches to the remodeling activity of the enzymes, unlikely previous conceptions of a more static and internalized behavior of these branches. As discussed next, the spin relaxation measurements of these branches have ultimately confirmed these dynamic properties .
NMR resonance linewidths (half-height of the peak, in Hz), which are directly related to the transverse relaxation rates (R2 values) are values indicative of dynamic properties. Narrow lines indicate decreased relaxation rates and thus increased rates of rotational motions. The linewidths of the 13C2-labeled galactose in the α1-6Man branch was over three-times that of the corresponding resonance from the α1-3Man branch (Table 3) . This suggested that the α1-6Man branch is considerably more immobilized than the other antennary. At experiments undertaken at higher fields, the 13C linewidths of the α1-6Man 13C2 resonance of galactose gave values more than three-times than the correspondent on the other branch. Interpretation using an isotropic model gave an effective correlation time of 37 ns, which is considerably longer than the hydrodynamic predictions for the Fc fragment, and the protein tumbling time (~20 ns). This suggested that although the α1-6Man branch is partially immobilized, there should be still some dynamic properties of this branch to explain the higher experimental value. Therefore a more complete set of relaxation time was measured at two different magnetic strengths (Table 3). A field-dependent R2 values were obtained, with smaller relaxation at 14.0 T (600 MHz) as opposed to 21.1 T (900 MHz). All R values combined at the lower magnetic field were not consistent with prediction based on isotropic tumbling model and have suggested that if the relaxation were solely dipole in origin, R2 would be even less at this field strength . Both the field dependence and these inconsistencies have suggested contribution from chemical exchange contributions to R2 relaxation mechanisms that originated from the α1-6Man branch sampling multiple conformational states on the microsecond to millisecond timescale and therefore, modulating chemical shift in this process. At least, some of these states have to have substantial internal motion to raise the rotational R1 value (R1p).
In order to probe the existence of chemical exchange contribution, relaxation dispersion experiments can be employed, in which transverse relaxation rates (R2) can be measured using Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences . Through this NMR experiment type, resonances that experience multiple chemical environments on a timescale near to νCPMG (variable pulsing rates due to varying delays between the forming 180o pulses of CPMG pulse sequences) must become more intense as a function of increasing pulsing rates. Results from this experiment using both α1-3Man and α1-6Man branches of IgG Fc with 13C-labeled galactose residues at 50o C, and at two different magnetic strengths (21.1 T, 900 MHz, and 18.8 T, 800 MHz) have shown this profile (Figure 9). At low pulsing rates, the relaxation measurements approach predictions using linewidths. At higher pulsing rates, chemical shifts are canceled out due to rapid chemical shift refocus, therefore chemical exchange contributions can be sorted out. Clearly, the R2 values of α1-6Man branch decrease with pulsing rates, whereas the values for the other branch have kept constant (Figure 9A). The change in R2 in the presence of chemical exchange between two states (A and B) was described using the equation R2(1/τcp) = R20 +
The effects of chemical exchange rates on R2 can be also investigated using the rotational spin lattice-relaxation (R1p) measurements. R1p of both branches (Table 3) approach the rapid pulsing limit of R2 in CPMG experiments (Figure 9), which represents the approaching to a non-exchange contribution to R2. The large deviation seen for the α1-6Man branch is consistent with the considerable chemical exchange contribution. Taking all these relaxation-based data, two conformational states were observed for α1-6Man branch: one related to chemical exchange contribution within contact with the peptide chain, and another regardless the molecular contact resulted from low chemical exchange contributions . Taking together the existence of these both states, a dynamic behavior for the α1-6Man branch has been proved, while the constant low chemical exchange contribution of α1-3Man branch has pointed towards a glycan segment more externalized from the Cγ2 domain (Figure 8). These conclusions will be sustained by other results about chemical shift changes , as described in the following item.
2.4. Applications and implications of chemical shifts in glycobiology
Chemical shift values (δ, in ppm) together with coupling constants comprise the major information extracted in NMR studies of proteins and nucleic acids , and thus are of great usefulness in glycobiology as well. Chemical shift values provide reliable information concerning the chemical environments in which a given atom (nucleus) molecularly experiments at certain timescale. If there were no other kinds of interactions in addition to the Zeeman interaction  during the NMR experimentation, all nuclei of a given molecule would lead to the same frequency in a 1D NMR spectrum. Zeeman states represent the two spin possibilities of a spin-half nucleus (usually defined as α- and β-spin states) under the static magnetic field (B0) . Due to changes on the chemical environment of the nucleus within the molecules chemical shifts may experience different values which will result different frequencies even though the same isotope and with same Larmor frequency. One of the major determinants in the chemical shifts is the electronic shielding generated by the electrons that surround the spin-half nuclei. Electrons at these surrounds have also angular momentum and thus magnetic moment. Differential conformational states may give different chemical shift values in a limited time-range. This can be explored to understand dynamic behaviors of glycans and to understand the chemical structures of biomolecules. Another application in the exploration of chemical shift changes is in glycan-involved intermolecular complexes, such as those of carbohydrate-binding proteins. The former phenomenon will be briefly explained through the dynamic properties of the α1-6Man branch of IgG Fc , continuing the same example taken at the previous section. The latter phenomena on concerning intermolecular complexes involving carbohydrate-binding proteins and glycans as common ligants will be discussed afterwards, taken some unpublished data about the binding properties of the chemokine RANTES complexed with chondroitin sulfate hexaccharides of well-defined sulfation patterns.
2.4.1. The dynamic behavior of glycans through chemical shift changes: the bound and unbound states of the N-glycan of IgG Fc
The chemical shifts of the Fc α1-6Man galactose residue were observed to be clearly temperature-dependent (Figure 9B). The 13C2-resonance can show a significant displacement between 5-50oC with its 13C-chemical shift trending toward a plateau of 75.3 ±0.2 ppm at the high-temperature limit (Figure 9B) . This plateau value is quite consistent with chemical shift values from the free glycan in solution which implies that the glycan is far away from chemical environment promoted by the Fc amino acids (state A, Figure 10). Although Figure 9B showed a curve-fitting only for the C2 resonances, the 13C6-chemical shift showed a similar trend and moved from a perturbed resonance position at lower temperatures towards a free glycan position at higher temperatures .
Through the use of the chemical shift values determined for state A in conjunction with the observed chemical shifts at 50oC obtained from CPMG relaxation dispersion data, the 0.8 value was extracted for the state A population (PA) using the equation PA = 1/[ω02(δX-δA)2/
2.4.2. Unveiling binding sites of carbohydrate-binding proteins through chemical shift perturbation: The chemokine CCL5/RANTES-chondroitin sulfate hexasaccharides complexes
Chemokines are a family of small cytokines with chemotactic properties. They are small and soluble proteins (with a mass of 8-16 kDa), and are produced and released by a variety of cell types during the initial phase of host responses to injury, allergens, antigens, or invading microorganisms. Chemokines share a sequence homology and possess four cysteines in conserved locations. These form two disulfide linkages that are keys to their tertiary structure and stability (Figure 11). These cysteines are also involved in the nomenclature and classification of the chemokines. Among many chemokines, CCL5, also known as RANTES, is a 68-residue proinflammatory chemokine responsible to control migration and trigger activation of leukocytes during their trafficking to the inflamed sites. Both these biological activities of CCL5 have shown to be critically influenced by interactions with GAG in endothelial surface proteoglycans . The immobilization of the CCL5 onto surface proteoglycans will attract leukocytes to the sites of injury, helping the rolling step which is heavily driven by selectin-mediated interactions; followed by the activation step of the leukocytes. This step is driven by the interaction of the CCL5 receptor on the leukocyte (CCR5) with the immobilized CCL5. GAGs in proteoglycans do not only regulate these two steps but also induce the oligomerization of CCL5 . This GAG-induced oligomerization process is crucial to i) create a high local concentration of chemokine to optimize the chemoattraction of cell for sites of lesion, ii) increase the
The 15N-HSQC spectrum of the 15N-labeled E66S CCL5 (Figure 12), which is mostly found as monomers in solution rather than the oligomerization propensity of the wild type , showed well-resolved amide signals that allowed easily a near-complete chemical shift assignment (Table 4). These initial assignments are crucial to further inform the chemical shift changes induced by the presence of increasing concentration of the ligants. The ligants used were two chondroitin sulfate hexasaccharides fully characterized by NMR . They are named CS 6-6-4, and CS 4-4-4, which represent the following respective structures: ΔUA(β1→3)GalNAc6S(β1→4)GlcA(β1→3)GalNAc6S(β1→4)GlcA(β1→3)GalNAc4S-ol, and GlcA(β1→3)GalNAc4S(β1→4)GlcA(β1→3)GalNAc4S(β1→4)GlcA(β1→3)GalNAc4S-ol. The abbreviations are ΔUA for Δ4,5unsaturated uronic acid; GalNAc for N-acetyl galactosamine; GlcA for glucuronic acid; “S” for sulfation group, digits before “S” represent the ring position; and -ol stands for reduced sugars (open rings at the reducing-end terminal units) .
As the concentration of the chondroitin sulfate ligants increases, either loss of signal intensity or chemical shift migration were observed on the 15N-HSQC spectra of E66S CCL5 (Figure 13). In the case of the continuous titration using the ligant CS 6-6-4 (left-hand side spectrum of Figure 13), the signal intensity decreases proportionally, and no chemical shift migration was seen. This likely indicates a slow exchange rate between the on- and off-states of the complex, and the GAG-induced oligomerization was observed since large amounts of precipitates were visually formed on the bottom of the NMR tube during the titration experiment (data not shown). This precipitation phenomenon is one of the major reasons for the NMR signal intensity loss. Conversely, the titration using the ligant CS 4-4-4, no intensity loss was observed even using 10 equivalent molar of this ligant (blue spectrum), and clear chemical shift migration of certain peaks was observed (right-hand side spectrum of Figure 13). In the experiment using this ligant, the amino acids that showed the major chemical shift changes were S1, Y3, D6, T7, R17, K45, N46, R47, Q48, as indicated in the spectrum of Figure 13. The residues K45, N46, and R47 match perfectly with the heparin binding motif (Table 4) . These residues that experience the largest chemical shift migration are highlighted on the structure of CCL5, represented in both cartoon and surface structural models (Figure 14). They are located at the pocket of the dimer consisted of the 40s loop, which in turn includes the heparin binding motif, together with the N-terminal (Figure 11).
In order to understand the physiological condition of the GAG-induced oligomerization of CCL5 promoted by the CS 6-6-4, and the nature of the binding involved in the interaction between these two molecules, an experiment using increasing concentrations of sodium chloride was performed (Figure 15) at the 2-times molar excess of ligant. This was the condition that showed the huge precipitation and loss of signal intensity (black spectrum at left panel of Figure 13). It is clearly observed that at physiological salt concentration (150 mM NaCl) the low intensity of CCL5´s signals still indicate oligomerized states of the chemokine. However, this highly-complexed state is broken down at higher salt molar concentrations than the physiological condition as seen with the recovery of the signal intensity in the spectrum with 300-450 mM. This result indicates that only at higher salt concentrations than the physiological condition, the oligomerization of CCL5 is abrogated and the nature of the binding between the two molecules is just electrostatic. The return in loss of the signal intensity in the 15N-HSQC spectra of CCL5 is just consequence of an instrumentation limitation due to the extreme high salt molarity that disturbs the magnetization sensitivity on the probe (the detection devise of the magnet). In synthesis, the CS 6-6-4 induces CCL5 aggregation whereas CS 4-4-4 just a little. This preliminary result indicates that the oligomerization of this chemokine is triggered by specific regions of the GAG chains in endothelial surface proteoglycans.
2.5. Scalar and dipolar coupling constants in glycobiology NMR
Besides chemical shifts, coupling constant values, either scalar (
2.5.1. Scalar couplings in conformational analysis of sugar rings
In general, three chemical bond proton-proton (3
|α-IdoA 4C1 (calc)||2.0||2.1||2.0||2.0|||
|α-IdoA 2S0 (calc)||4.3||11.1||5.4||5.4|||
|α-IdoA 4C1 (calc)||1.7||3.0||3.1||2.5|||
|α-IdoA 2S0 (calc)||4.8||10.5||3.4||2.5|||
|ΔHexA 1H2 (calc)||3.0||1.5||4.1||4.1|||
|ΔHexA 2H1 (calc)||8.3||7.7||2.8||2.8|||
|ΔHexA 1H2 (calc)||3.1||0.2||6.0||nd|||
|ΔHexA 2H1 (calc)||8.3||10.2||1.7||nd|||
The conformation set observed for iduronic acid (IdoA) units (Tables 5 and 6), is complex and seems to be influenced not only by their sulfation patterns but also by the adjacent residues in which the IdoA units are linked to. For example, in case of IdoA units of a dermatan sulfate-tetrasaccharide studied at reference , the conformer population was estimated through Karplus relationship, and showed the 80/20 ratio for the chair 4C1 and skew-boat 2S0 conformations in solution. These IdoA units are linked to GalNAc units in dermatan sulfate, and are not sulfated. But when 2-sulfated or linked to GlcNAc units, as shown in other works presented at Table 6, different population ratios were observed, including the chair 4C1 conformer together with the chair 1C4, and the skew-boat 2S0 conformers (Figure 16). In a heparin-derived tetrasaccharide, the chair 1C4 and skew-boat 2S0 conformer population ratios changed slightly for 76:24% (Table 5) . The half-chair 1H2 and 2H1 populations of the unsaturated uronic acid in this heparin-derived tetrasaccharide changed to the 78:22% ratio  rather than 100% 1H2 conformer population for Δ4,5HexA in a dermatan sulfate-derived tetrasaccharide  (Table 5) due to the presence of the adjacent GlcNAc unit rather than the GalNAc unit. Therefore, IdoA units can experience different ring conformer population ratios based on the neighboring units (Tables 5 and 6).
2.5.2. Dipolar couplings in conformational studies of oligosaccharides
Dipolar couplings (D) arise from through space spin-spin interactions and are strictly dependent on both inter-nuclear distance (r) and the angle (
Evidences of spatial structures of oligosaccharides are gradually increasing along the past few years with the advent of specific NMR methods and their application in glycobiology. Although NOE seemed the primary choice in conformational NMR studies, sometimes in the analysis of oligosaccharides the NOE-signal intensity that relies on the efficiency of polarization transfer between proton pairs may go to zero or close to zero because of the correlation time dependence cross-relaxation. In addition, the usual high-order flexibility of oligosaccharides, as opposed to a more rigid structural behavior of nucleic acids and proteins, may result NOE-contacts only from few conformers experienced within a short timescale. This ultimately lowers the efficacy of the NOE-based method in carbohydrate studies. Moreover, while NOE-based structural determination is basically based on through space inter-proton physical contacts, theoretically all types of nuclei pair combinations of spin vectors (1H-1H, 13C-1H, 15N-1H, 13C-13C) can be evaluated by the RDC method. Therefore, RDC-based measurements become the alternative NMR method in studies of oligosaccharides, not only on the conformational perspective but also in dynamic analysis. Using residual dipolar couplings (RDC), many carbohydrates have been characterized by the conformational perspective [42, 43, 48]. Here, we briefly revised the conformational and dynamical view of a heparin-tetrasaccharide [ΔUA2SO3-(1→4)-GlcNS,6S-(1→4)-IdoA2S-(1→4)-GlcNS,6S], notated as D-C-B-A, studied by RDC . And in this study, a limited flexibility of the IdoA-composing unit of this tetrasaccharide was unexpectedly observed through the dynamic point-of-view.
Table 7 summarizes experimental 1
In order to obtain the order tensor parameters, the analysis was divided into two sets depending on the conformation of the ring B, and thus, their diagonalized order tensors were inspected. The average Saupe matrix, mostly utilized to build up the molecular frame in RDC-based studies , of the 1C4 form was found to have principle axis components:
2.6. NOE in glycobiology NMR
The nuclear Overhauser effect (NOE)-based NMR studies have been considered the foundations of biomolecular NMR not only through the conformational perspective but also useful in deciphering the hydrogen-bond networks of intermolecular complexes containing glycans. This was the main technique applied by the Nobel Prize-awarded group of Kurt Wuthrich in structural studies of biomolecules by NMR spectroscopy. NOE-signals result from a relaxation phenomenon and provide a through-space distance between two nuclei (usually protons) from the same molecule (intra-molecular NOE) from the same residue (intra-residue NOE) or those induced in the presence of binding residue (inter-residue NOE), as well as from different molecules (inter-molecular NOEs). The latter is usually studied by the specific transferred NOE (trNOE)-signals . Next, we will give few brief examples in glycobiology successfully using these two NOE-related NMR techniques.
2.6.1. The classical through space NOE-based structural and conformational studies
As already documented in item 2.5.1, IdoA units can experience in solution the chair 1C4, and 4C1 as well as the skew-boat 2S0 conformations. The relative proportion of each conformer population varies as function of the adjacent units and attached substituents (if any) . The contribution of each of these conformation populations can be determined by 3
Unlikely the previous example of intra-residue NOE-signal, in the work of Castro et al. , an intramolecular inter-residue NOE-signal has been assigned between the (1→3) glycosidic bonds of β-galactosyl units in a marine sulfated galactan. This NOE-signal was crucial to define the repetitive disaccharide unit of this new structure of sulfated glycan .
NOE-signals are the most important NMR information for conformational studies, including of carbohydrates. The existence of conformational structures of some oligosaccharides in solution can be seen by the examples of tri-, tetra-, and pentasaccharides of (1→2)-β-mannopyranosyl units studied by NOESY (nuclear Overhauser effect spectroscopy), and ROESY (rotational frame nuclear Overhauser effect spectroscopy) . These both 2D NMR spectral types are capable to display cross-peaks from through space 1H-1H connections, however, with different intensities as a function of the molecular size or correlation-time. ROE-signals are always positive, ranging from ~40 to 65% intensity, while NOE-signals has larger range, from 50% positive peak intensity to 100% negative peak intensity, according to the molecular size and correlation-time . Therefore, as NOE- and ROE- resonances depend on the size of the molecules, both experiments were recorded for the mannopyranan oligosaccharides . Inter-proton distances were calculated based on the r-6 relationship between distance and NOE/ROE cross-peak intensity related to a signal reference from the same studied molecule with known parameters. In this example case, the intra-residue H1-H5 contact, set to 2.39 Å was used found on relevant crystal structure models , and the values obtained are displayed in Table 8.
|Contacts across glycosidic linkage||observed||calculated||observed||calculated||observed||calculated|
|Contacts separated by one residue|
|Contacts separated by two residues|
All the inter-glycosidic contacts (H1-H2) between linked mannosyl units are assigned and with similar distance values (Table 8). The rising numbers of other spatial contacts in tri-, tetra- and pentasaccharide, point clearly towards defined 3D conformers of these molecules. The presence of contacts between H4 with H1 and H2 of residues located two units away in a chain, strikingly supports the presence of ordered structures of these oligosaccharides in solution .
2.6.2. Protein-bound carbohydrate conformations seen by transferred NOE
Transferred NOE (trNOE) experiments can reveal the bioactive conformations of protein-bound carbohydrates [56-59]. This is very important in glycobiology since the recognition of binding conformers of sugars to enzymes, antibodies and lectins are of great interest in glycobiology, especially for in carbohydrate-based drug design. trNOE is obtained from a regular NOESY experiment although at a protein-carbohydrate mixed samples that posses some dynamic exchange. The carbohydrate ligand must be in excess of the protein, and thus trNOE can be collected from the free ligant that experience the “on-state” upon physical contact with the protein. In complexes involving large molecules, cross-relaxation rates of the bound state (
The use of trNOE can be illustrated by the work concerning the protein-bound conformational characterization of the mimetic ligand β-D-Glc
3. Marked conclusions
Despite the recent association of NMR with glycobiology, relevant results have ultimately appeared. Although carbohydrates posses high-order degrees of flexibility, and usually great structural complexity; NMR methods still seem quite able to elucidate the main structural characteristics and dynamic behaviors of the majority of glycans. Since NMR spectroscopy is currently the most advanced and powerful structural technique, despite its sensitivity issue, its contribution to the glycobiology’s progress, and thus to the current glycomics’ is profound. New NMR methods have been adjusted just for carbohydrate analysis, inclusively specific isotopic labeling protocols to overcome the sensitivity problem. Proton, carbon-13, nitrogen-15 by either one- or multi-dimensional NMR experiments, chemical shifts, scalar coupling constants, dipolar coupling constants, and NOE-through space connections of free or protein-bound carbohydrates comprise the principle NMR spectroscopy methods for glycobiology. Many other NMR techniques, such as saturation transfer difference and modified pulse sequence destined just to address glycobiology-related problems do exist, although not covered in this chapter. The main idea of carbohydrates as just energetically or structurally involved-molecules, has falling apart as many other vital functions of glycans, mostly in signaling events, have been unraveled along the past few years. And NMR spectroscopy is making an outstanding contribution for these big discoveries.
The author acknowledges InTech-Open Access Publisher for the kind invitation in contributing with this chapter; Dr. John Glushka to the help on the GlcNS assignment; Dr. Ana Paula Valente to the help on the 13C-direct observe spectrum of Glc, and the accessibility to the Bruker 400 MHz spectrometer; and Prof. James H. Prestegard for all NMR background during my post-doctorate period, and the accessibility at the Varian 800 MHz spectrometer. All the data discussed in this chapter in which the Varian 800 MHz is indicated in figure legends, were recorded during the author´s post-doctorate period at Complex Carbohydrate Research Center, the University of Georgia, US under the partial financial support from the National Center for Research Resources of the National Institute of Health, RR005351, and from the post-doctoral fellowship (PDE #201019/2008-6) from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.