Direct Observation of Carbohydrate Hydroxyl Protons in Hydrogen

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Direct Observation of Carbohydrate Hydroxyl Protons in Hydrogen Bonds with a Protein Gustav Nestor, Taigh Anderson, Stefan Oscarson, and Angela M. Gronenborn J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10595 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 11, 2017

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Direct Observation of Carbohydrate Hydroxyl Protons in Hydrogen Bonds with a Protein Gustav Nestor,† Taigh Anderson,‡ Stefan Oscarson,‡ and Angela M. Gronenborn*,† †

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, United States ‡

Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland

ABSTRACT: Hydroxyl proton resonances of uniformly 13C-labeled Manα(1-2)Manα(1-2)ManαOMe (Man3) bound to cyanovirin-N (CV-N) were detected at ambient temperature in aqueous solution by NMR spectroscopy. The directions of the hydroxyl groups were determined based on NOEs and a previously unknown hydrogen bonding network between Man3 and CV-N was discovered. This is the first report on detecting hydroxyl protons of a protein-bound carbohydrate in aqueous solution by NMR. Approaches such as presented here may open the door for accurately determining inter-molecular hydrogen bonds in carbohydrate-protein complexes.

INTRODUCTION Hydroxyl groups are prevalent in carbohydrates and mediate important molecular properties as well as hydrogen bonds. Hydrogen bonding plays a critical role in carbohydrate recognition by lectins, although its direct observation by NMR is still lacking. This is in contrast to nucleic acids and proteins, for which inter-residue hydrogen bonds involving imino, amino, amide and hydroxyl protons can be detected by NMR via through-bond couplings.1-5 For carbohydrates, only intra-molecular hydrogen bonds have been identified to date via through-bond couplings by NMR.6-8 Unless neutron9 or very-high resolution X-ray diffraction data10 are available, hydrogen atoms are not observable by crystallography. The position of protein amide backbone protons commonly is inferred from the positions of the nitrogen atoms in the polypeptide backbone. Hydroxyl protons in sugars, by contrast, are difficult to position since they occupy different positions in the three different rotamers (trans, gauche+ and gauche-), given the free rotation of the C-OH bond. In general, hydroxyl proton hydrogen bonds inferred from X-ray crystal structures are predominately based on the spatial relationship between the hydroxyl oxygens and possible hydrogen bond acceptors nearby. However, this qualitative approach entails the risk that, in reality, the hydroxyl proton may be pointing into a different direction. Spectroscopically, hydrogens are the most common atoms observed in NMR spectra, although for exchanging protons such as imino, amide, amino and hydroxyl protons, chemical exchange with water often prevents their detection, unless the exchange is relatively slow. This is the case for amino and amide protons. Hydroxyl protons, however, exchange fast with water protons and are only observable if the exchange rate is slowed by hydrogen bonding or other protection. Indeed, in protein NMR spectra, hydroxyl protons

from serine, threonine and tyrosine are observable when involved in hydrogen bonding.4, 11-12 In the NMR spectra of RNA, the ribose 2′-hydroxyl protons are observable and their preferred orientations have been thoroughly evaluated using coupling constants and NOEs.13-19 Carbohydrate hydroxyl protons are ordinarily not detected by 1H NMR spectroscopy, unless very high concentrations and/or sub-zero temperatures are used for slowing down exchange. Frequently, low temperatures and the addition of 10-15% organic solvent, such as acetone, methanol, or DMSO to prevent freezing of the sample is employed.20 Carbohydrate hydroxyl protons in complexes between galactosides and lectins or antibodies in fast exchange have been observed in mixed solvents (water/DMSO),21-22 although effect(s) of the aprotic solvents on protein conformation and ligand binding renders interpretation fraught with difficulties. Indirectly, proteinbound carbohydrate hydroxyl protons can be identified via cross relaxation with non-exchangeable protons using water-selective NOESY-HSQC experiments, although a direct hydroxyl proton resonance is not observed in such experiments.23 To the best of our knowledge, carbohydrate hydroxyl protons have never been observed in any NMR spectrum of a carbohydrate-protein complex in aqueous solution up to now. We previously reported that isotope-edited NOESY experiments can be used to simultaneously determine contacts on both, the protein-bound carbohydrate and the protein, using 13C-labeled sugars and 15N-labeled proteins.24 Here, for proof of concept, we used uniformly 13 C-labeled Manα(1-2)Manα(1-2)ManαOMe (Man3; Figure 1a) bound to 15N or 13C/15N-labeled cyanovirin-N (CV-N), and detected four carbohydrate hydroxyl protons. Their resonances, surprisingly, were visible in the room temperature spectra of the complex and were assigned via scalar couplings from the adjacent sugar ring protons.

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Intra- and intermolecular NOEs involving these hydroxyl protons permitted the determination of their orientation and hydrogen bonding patterns. Our results augment currently available structural data on the Man3-CV-N complex and, in general, set the stage for characterizing hydrogen bonding in other carbohydrate-protein complexes.

Figure 1. (a) Structural formula of Man3 (Manα(1–2)Manα(1– 2)ManαOMe). The experimentally observed hydroxyl protons in the Man3/CV-N complex are shown in red. (b) Newman projections of Man3 hydroxyl proton rotamers (OH3, OH4, OH3ʹ, and OH4ʹ), depicting the hydroxyl oxygen free electron pairs as orbital lobes with those accepting H-bonds marked with asterisks. The carbon atom is positioned at the front and the hydroxyl oxygen at the back. (c) Superposition 1 13 of H, C-CT-HSQC-TOCSY (blue, 20 ms mixing time) and 1 13 13 H, C-CT-HSQC spectra (red) of C-labeled Man3 bound to CV-N recorded at 30 °C. Correlations between hydroxyl protons and adjacent sugar ring protons are connected by dashed lines.

spectrum may arise from differences in exchange rates, with faster exchange resulting in lower intensity, or to differences in 3JHCOH for the different positions in the sugar rings. Given the data and analysis of the 1H,13CHSQC-NOESY spectra, the different intensities in 1H,13CHSQC-TOCSY spectrum have to be related to differences in 3JHCOH, and, by inference, differences in torsion angle. In general, averaged 3JHCOH values are ~5.7 Hz,25 and given the fact that different HSQC-TOCSY cross-peak intensities were observed here, restricted rotation around the COH bond is the most likely cause. Based on the 3JHCOH Karplus curve parameterized for carbohydrates, the trans, gauche+ and gauche- rotamers (Figure 1b) will exhibit large couplings (>10 Hz) for the trans rotamer, while the gauche rotamers will possess small couplings (~1 Hz). Although accurate values for the vicinal coupling constants could not be measured for the complex, given the broad lines, large (>10 Hz) and small (~1 Hz) coupling constants will by necessity translate into different intensities of the HSQC-TOCSY cross-peaks (Figure 1c). We therefore suggest that the OH3 and OH3′ resonances, which exhibit intense TOCSY cross-peaks, are associated with the trans rotamer, while OH4 and OH4′, which exhibit weak cross-peaks, arise from the gauche rotamer. The four sugar hydroxyl proton resonances were detected at all examined temperatures (5-31 °C), and their intensities increased at lower temperatures. Since the overall spectral quality is superior at higher temperatures (20-31 °C), with narrower lines for both sugar ring and protein protons, the majority of experiments were performed at 20 or 30 °C. Note that the strip that contains the OH3′ resonance in the 1H,13C-HSQC-NOESY spectrum also includes the T7 hydroxyl proton resonance of the protein and therefore, NOEs between the threonine hydroxyl proton and sugar ring protons are also visible (Figure 2).

RESULTS AND DISCUSSION Observation and assignment of hydroxyl proton resonances. Hydroxyl proton resonances were observed in 1 H,13C-HSQC-TOCSY spectra of CV-N-bound Man3 (molar ratio 1:1; 0.8 mM) and assigned via three-bond scalar coupling correlations (3JHCOH) to the vicinal ring protons (Figure 1). For larger mixing times (>20 ms), additional correlations to more ring protons within the same ring can be observed, validating the assignment. We detected the OH3 and OH4 hydroxyl proton resonances of the reducing-end sugar, and the OH3′ and OH4′ resonances of the middle sugar in Man3. In the 1H,13C-HSQC-TOCSY spectrum, the correlation cross-peaks involving OH3 and OH3′ were much stronger than those of OH4 and OH4′. Such different intensities in the 1H,13C-HSQC-TOCSY

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Figure 2. Selected strips from H, C-HSQC-NOESY spectrum recorded at 20 °C (60 ms mixing time), illustrating intramolecular NOEs involving sugar hydroxyl protons, and NOEs between sugar ring protons and T7 OH.

Chemical shifts and temperature coefficients. All chemical shifts and temperature coefficients for the OH protons are summarized in Table 1. Comparison of the OH chemical shifts with those reported for free Man3 by Hakkarainen et al,26 reveals that the OH3 resonance shifts downfield (0.9 ppm) upon binding to CV-N, while the OH4 and OH3′ resonances experience upfield shifts (-1.6 and -0.6 ppm, respectively). For the OH4′ resonance, on the other hand, no significant chemical shift changes were noted between free and CV-N-bound Man3. It is generally assumed that hydrogen bonding results in a downfield shift, i.e. deshielding the nucleus that acts as a H-bond donor. No systematic studies, however, have been performed for carbohydrate OH protons in proteinsugar complexes. In free sugars, the only example of a downfield shift (>1 ppm), to the best of our knowledge, has been reported for a hydroxyl proton which is involved in an intramolecular hydrogen bond between the OH and a phosphate group in myo-inositol compounds.27 Scrutinizing the data available for threonine and serine OH protons revealed that they resonate over a very broad range of chemical shifts (2.9 to 7.8 ppm), and no discernable dependence on H-bonding was apparent.11 Therefore, interpretation of chemical shift differences upon hydrogen bonding of OH protons, whether for carbohydrates or amino acid side chains, appears to be complex and needs further in-depth theoretical and experimental investigation. Table 1. Summary of hydroxyl proton properties.

δH (ppm) a

Δδ

dδ/dTbound dδ/dTfree

θHCOH

b

Rotamer 3

JHCOH

a

b

c

HBAcceptor rOH-A

d

θOH-A

OH3'

OH4'

T7 OH

6.78

4.36

5.27

5.82

5.32

0.9

-1.6

-0.6

-0.1

-

-5.8

-3.9

-3.4

-1.2

-3.7

-11.4

-10.2

-10.7

-11.0

-

150

75

160

-60

165

+

t

g

-

t

-0.6

12.9

1.3

-

N93 C'O E23 C'O G2 C'O K3 C'O

OH4'

t 10.9

g

2.43

2.16

1.87

1.41

1.91

134

154

149

131

158

d

141

142

151

132

-

HBDonor d d

θDH-O a

OH4

d

θC′O-H rD-OH

OH3

D95 NH

N93 NH T7 OH N93 δNHb

2.17

1.71

1.91

1.91

166

170

158

175 26

δH and dδ/dT of free Man3 are from Hakkarainen et al. Chemical shifts of free Man3 measured at -10 °C in 85% H2O/15% acetone-d6, were extrapolated to 30 °C. Δδ = b δ(bound) - δ(free). OH dihedral angles were determined c from intramolecular OH-CH NOEs (see Table 2). Coupling

constants (Hz) were calculated from the Karplus relation3 ship: JHCOH = 5.76 – 2.05 cos θ + 6.78 cos (2θ), parameterized 25 d by Zhao et al. Extracted from the X-ray structure model (PDB accession code 3GXZ) after adjustment of the carbohydrate glycosidic angles and OH dihedral angles based on the NMR data.

Temperature coefficients (dδ/dT) for the OH proton resonances were measured from 1H,13C-HSQC-NOESY spectra over the temperature interval from 5 and 31 °C (Figure S1). They ranged from -1.2 ppb/K for OH4′ to -5.8 ppb/K for OH3 (Table 1). In general, chemical shifts of exchangeable protons, which are involved in hydrogen bonding or otherwise protected from exchange, exhibit small temperature coefficients, compared to solvent exposed protons. However, all conformational changes will contribute to the chemical shift and in proteins, temperature-induced structural changes also contribute to the temperature coefficients of amide protons.28 For entirely solvent exposed carbohydrate OH protons, dδ/dT values of ~ -11 ppb/K have been reported, which were also observed for free Man3.26 Given the smaller values measured here for CV-N-bound Man3, these sugar hydroxyls are clearly less solvated, consistent with the fact that their resonances are readily observed at room temperature. Of all four hydroxyls identified and observed by NMR, the OH3 sits at the outer edge of the sugar binding pocket, in agreement with a more pronounced temperature dependence of its hydroxyl H resonance. The very small temperature dependence of the OH4′ hydroxyl resonance (dδ/dT = -1.2 ppb/K) is similar to dδ/dT values of CH ring proton resonances, which range from -1.6 ppb/K (H1′) to +1.8 ppb/K (H4″). Therefore, the temperature dependence of the OH4′ hydroxyl proton resonance may be caused by a conformational change in the protein and not a change in water exchange. Intramolecular NOEs permit the determination of O-H group directionality. The extraction of precise distance information from OH proton NOEs is fraught with difficulties given the pronounced chemical exchange between OH protons and water, which dramatically influences cross-peak relaxation. In order to handle this problem, full relaxation matrix analysis has previously been employed.29 However, the OH protons observed here exhibit slow exchange with water and negligible waterexchange cross-peaks are present in the NOESY spectra. We therefore used the NOEs in a qualitative fashion, without taking water exchange into account. In our previous work on the CV-N/Man3 complex, distance information between carbon-attached sugar ring protons and between these sugar ring protons and protein protons were extracted from 2D 1H,13C-HSQC-NOESY spectra .24 Here we utilized the same series of 2D 1H,13CHSQC-NOESY spectra (Figure 2) for a number of mixing times (10-120 ms) in order to construct NOE build-up curves from cross-peak intensities involving hydroxyl protons (Figure S2a), and extracted distances from the initial slope of the build-up curves. Three to four distances were obtained for each OH proton (Table 2 and S1), which permitted the determination of H-C-O-H dihedral



angles, θHCOH, by comparing the experimental values with model-derived distances. The molecular model was constructed by adding protons to the coordinates of the crystal structure of the CV-N/Man-9 complex (PDB accession code 3GXZ), and dihedral angles for each OH hydroxyl proton were varied in 30° increments, with distances from these model structures compared to the NOE-derived distances (Figure S3). For the angle that appeared close to correct, further refinement was carried out using 5° increments around the initial optimum. The final optimized conformations (Table 1) show that the OH3 and OH3′ hydroxyl protons are in the trans conformation (150° and 160°, respectively), whereas OH4 and OH4′ hydroxyl protons are close to gauche (75°, g+ and -60°, g-, respectively). These dihedral angles are in excellent agreement with the qualitative interpretation of the 1H,13C-HSQC-TOCSY spectrum (see above). A parameterized Karplus relationship, derived by Zhao et al. for hexapyranosides,25 was used to calculate 3JHCOH values from dihedral angles (Table 1), and gratifyingly explains that the more intense cross peaks for OH3 and OH3′ in the 1H,13C-HSQC-TOCSY spectra are related to the larger vicinal 3JHCOH values. Table 2. Intramolecular NOE-derived distances (Å) between Man3 OH and sugar ring protons, compared to distances derived from an X-ray structure (PDB code 3GXZ) after addition of hydroxyl protons based on the dihedral angles in Table 1. rexp

a

a

r3GXZ

OH3

H2

2.69

2.67

OH3

H3

2.74

2.77

OH3

H4

2.59

2.63

OH4

H3

3.14

3.57

OH4

H4

2.40

2.38

OH4

H5

2.68

2.68

OH4

H6a/b

2.30

1.76

OH3′

H2′

2.80

2.91

OH3′

H3′

2.54

2.83

OH3′

H4′

2.42

2.51

OH3′

H1″

3.13

3.47

H4′

2.30

2.29

OH4′

H3′/5′

2.69

2.79

OH4′

H6′a/b

3.12

3.09

H6a/b- OH4 and H6′a/b- OH4′ distances (Table 2) depend on the orientation of the C6 hydroxymethyl group, which is defined by two ω angles (O5-C5-C6-O6 and C4C5-C6-O6). Intramolecular NOEs yield ωO5 = -50° for O6, close to a gg conformation, and ωO5 = +85° for O6′, close to the gt conformation (Table S2). However, contributions from other rotamers cannot be excluded and for a sugar in solution, the C6 hydroxymethyl group is generally treated as exhibiting conformational averaging between gt, gg, and tg rotamers. For free mannose, a 1:1 ratio for gt and gg rotamers is commonly assumed, with the tg conformer only present in very small amounts, given the repulsive interaction between O6 and O4.30 In the X-ray crystal structure of CV-N/Man-9, the gt rotamer is observed for O6 with the oxygen atom positioned for hydrogen bonding with E23 εO.31 This H-bond is only possible in the gt rotamer. Since we find O6 as the gg rotamer, based on NOE data, we suggest that the OH6 hydroxyl instead forms a hydrogen bond with N93 δN (2.8 Å O-N). For O6′, both the X-ray structure and the NOE data suggest a gt rotamer, with O6′ pointing away from the complex, not forming any direct hydrogen bonds. Rapid exchange between different rotamers and/or the lack of stable hydrogen bonds may contribute to the fact that OH6 and OH6′ resonances are not observed. We did not detect any resonances for the hydroxyl protons of the non-reducing end sugar. This is in good agreement with our previous data, which showed a flexible (1″→2′) linkage, compared to the (1′→2) linkage.24 We therefore conclude that any hydrogen bonding involving OH2″, OH3″, OH4″ or OH6″ hydroxyls is not persistent at the temperatures examined here.

b

OH4′

tance extracted from the X-ray structure may not be correct since the non-reducing end sugar does not possess the common chair conformation. In the solution complex investigated here, the combined NOE data, clearly shows the presence of 4C1 conformations for all three sugar residues.24

c b

b

Standard errors are 0.06 or less. The distance was calculated as a sum of the cross-relaxation contribution from H6a c (H6a′) and H6b (H6b′). The distance was calculated from OH4′ – H3′ (2.87 Å) and OH4′ – H5′ (3.52 Å).

Comparison of the NMR-derived distances with those deduced from the X-ray structure with hydroxyl protons in the optimized conformations are within ±0.2 Å, except for H3- OH4, H6a/b- OH4, H3′- OH3′, and H1″- OH3′. Among these, the H3- OH4 and H3′- OH3′ distances may be compromised by spin diffusion involving H4/ H5 and N93 NH, respectively. Furthermore, the H1″- OH3′ dis-

Intermolecular NOEs – OH-protein interactions. Intermolecular NOEs between sugar OH protons and protein amide protons were measured in 13C/15N-filtered NOESY-1H,15N-HSQC experiments for a sample of 13Clabeled Man3, complexed with 13C/15N-labeled CV-N (ratio 1:1, 800 µM). 13C/15N-filtering in f1 leaves hydroxyl and thiol protons unfiltered. Since CV-N does not contain any free thiol groups, the observed NOEs have to originate from either CV-N or Man3 OH protons (Figure 3a). NOE build-up curves were generated for a series of mixing times (20-100 ms; Figure S2b) and distances were derived from these. The NOE-derived distances were compared to those measured from the X-ray structure of CV-N/Man-9 with added OH protons in preferred conformations (Table 3). Only small differences of ±0.1 Å were noted between the NMR and X-ray data, except for OH4- N93 δNHa/b, OH4- G96 NH, and OH4′- T7 NH, where spin diffusion may play a contributing factor. Since we used Man3 in the current work and the X-ray structure contained Man-9 as the ligand, the presence of small struc-


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Journal of the American Chemical Society tural differences (±0.1 Å) between NMR and X-ray structure would not be surprising.

13

15

Figure 3. (a) Selected strips of a C/ N-filtered NOESY1 15 H, N-HSQC spectrum recorded at 20 °C (50 ms mixing time). Intermolecular NOE cross peaks between carbohydrate hydroxyl protons and protein amide protons are labeled by amino acid name and number and hydroxyl identity. (b) Illustration of hydrogen bond parameters used in the MS and listed in Table 1. D denotes a protein hydrogen bond donor.

tween T7 OH and one of the side chain amino protons of N93, together with intermolecular NOEs, were used to determine the orientation of the T7 hydroxyl group, which was found to be in trans, relative to T7 Hβ (Figure S3e). Hydrogen bonding network. To generate a model for the solution complex, hydroxyl protons were added to the trimannoside unit of Man-9 in the X-ray crystal structure of CV-N/Man-9 (PDB accession code 3GXZ) in a conformation that is compatible with the observed NOEs within the sugar. The resulting structure revealed a hydrogen bonding network in which the four sugar OH protons, for which detectable hydroxyl proton resonances were observed, point towards the G2 C′O, K3 C′O, E23 C′O, and N93 C′O protein backbone carbonyl oxygens (Figure 4). Hydrogen bond distances (O----H) range from 2.38 Å for OH3- N93 C′O to 1.41 Å for OH4′- K3 C′O. The determined hydrogen bond donor angles (O-H----O; see Figure 3b) lie between 131° (OH4′- K3 C′O) and 154° (OH4- E23 C′O) (Table 1), falling into the region of moderate hydrogen bonds (>130°), according to the classification by Jeffrey.32 The acceptor hydrogen bond angles (C′-O—H) are between 132° (OH4′- K3 C′O) and 151° (OH3′ – G2 C′O), consistent with the placement of the unpaired electron pair of carbonyl oxygens around ±120°.

Table 3. Intermolecular NOE-derived distances (Å) between Man3 OH and CV-N amide protons, compared to distances derived from an X-ray structure (PDB code 3GXZ) after addition of hydroxyl protons based on the dihedral angles in Table 1. CV-N

rexp

OH3

D95

2.3

2.4

OH3'

F4

3.8

3.9

OH3'

N93

2.3

2.2

OH4

T25

3.4

OH4 OH4 OH4

a

a

Man3

N93 δNHa

3.4

G96

Figure 4. Molecular model depicting the interactions between Man3 and CV-N that involve hydroxyl protons. The model is based on the X-ray crystal structure of CV-N in the presence of Man-9 (3GXZ), with hydroxyl protons added and positioned based on the NOE data. Hydrogen bonds are depicted by dashed lines.

3.4 b

2.9

b

4.1

b

3.7

2.6

N93 δNHb

r3GXZ

2.9

OH4

D95

3.6

3.6

OH4'

Q6

2.7

2.6

OH4'

T7

2.5

b

3.3

b

Standard errors are 100 ms for long-range HSQC) limit the chances to observe such couplings.

CONCLUSIONS This is the first report on direct detection of carbohydrate hydroxyl proton resonances in a sugar/protein complex by NMR. The orientations of four OH hydroxyl protons, OH3, OH4, OH3′, and OH4′ of Man3, bound to CV-N, which were observed in the complex investigated here, were determined based on intramolecular NOEs and TOCSY cross-peak intensities. These hydroxyls donate H-bonds to four different backbone carbonyl oxygens of CV-N. In addition, we also identified H-bonds between backbone amide protons and the mannose OH3 and OH3′ hydroxyl oxygens, as well as a hydrogen bond from the T7 side chain OH proton to the sugar OH4′ hydroxyl oxygen. This extensive interaction network is responsible for the specificity of Man3 binding to CV-N.

EXPERIMENTAL Carbohydrate synthesis. Uniformly 13C-labeled Manα(1–2)Manα(1–2)ManαOMe (Man3) was synthesized as described previously.24 Protein expression and purification. P51G CV-N, labeled with 15N or 13C/15N was expressed as described previously.24, 39 Samples for NMR were buffer-exchanged into 10 mM sodium phosphate buffer, 3 mM NaN3, 95/5% H2O/D2O, pH 6.6. NMR spectroscopy. NMR spectra were recorded over the temperature range 5-31 °C on Bruker 600, 700, 800, and 900 MHz AVANCE spectrometers, equipped with 5mm-triple-resonance, z axis gradient cryoprobes. Parame-


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Journal of the American Chemical Society ter settings for the NMR experiments are summarized in Table S4. Assignments of Man3 and CV-N resonances were obtained essentially as described before.24 CV-N carbonyl carbon resonances were assigned using a 3D HNCO spectrum. For hydroxyl proton resonance assignments of bound Man3, a sample of the 13C-labeled Man3/15N-labeled CV-N complex at 1:1 molar ratio was prepared at 0.8 mM. 2D constant-time 1H,13C-HSQC-TOCSY and 1H,13C-HSQCNOESY spectra were recorded to obtain complete assignments. Spectra were referenced to internal DSS (δH = 0.00 ppm, δC = 0.00 ppm). Temperature coefficients were extracted from a series of 2D 1H,13C-HSQC-NOESY spectra, recorded over the temperature interval of 5-31 °C. For temperature calibration, an external methanol standard was used. Intramolecular OH-CH cross-relaxation rates of CV-Nbound Man3 (with 13C-labeling) were measured at 20 °C and 900 MHz, using a 0.9 mM sample (1:1 molar ratio of CV-N and sugar). 2D 1H,13C-HSQC-NOESY experiments were recorded for ten different mixing times, ranging from 10 to 120 ms. In addition, a 3D NOESY-HSQC spectrum with Watergate water suppression was recorded on the same sample. This spectrum was used to measure the relaxation rate between H6a and H6b for distance calibration, using the H6a-H6b reference distance (1.78 Å). Measurements of intermolecular OH-NH crossrelaxation rates of CV-N-bound Man3 were carried out at 20 °C and 800 MHz on a 0.9 mM sample (1:1 molar ratio) of 13C-labeled Man3 and 13C/15N-labeled CV-N. 2D 13C/15Nfiltered NOESY-1H,15N-HSQC experiments were recorded for ten different mixing times, ranging from 20 to 100 ms. In addition, a 3D version of the same experiment was recorded on the same sample for assignment purposes. A 3D NOESY-HSQC spectrum with simultaneous evolution of 13C and 15N chemical shifts in t2 was used to obtain the relaxation rate between N93 δNHa and N93 δNHb for calibration of distances with respect to the NHa-NHb reference distance (1.77 Å). NMR spectra were processed with Topspin 3.1 (Bruker), and ccpNMR40 was used for resonance and NOE crosspeak assignments. From 2D 1H,13C-HSQC-NOESY spectra, each OH NOE cross-peak intensity was normalized with respect to the corresponding Man3 diagonal signal intensity in the same spectrum and NOE build-up curves were constructed using the PANIC approach, with linear fitting of the build-up curves.41-42 In the 2D 13C/15N-filtered NOESY-1H,15N-HSQC spectra, each NH NOE cross-peak was normalized with respect to the signal intensity of the corresponding amide proton in a 1H,15N-HSQC spectrum. NOE build-up curves were constructed and exponential fitting was used to obtain the cross-relaxation rates. Error values were obtained from the linear or exponential fitting of the NOE build-up curves. The isolated spin-pair approximation (ISPA) was used to extract distances relative to known reference distances. Experimentally determined distances were compared to the equivalent distances in the D1 arm trimannoside of

Man-9 extracted from the X-ray structure with CV-N (PDB accession code 3GXZ)31 with protons added using PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC). The glycosidic torsion angles equivalent to the (1′→2) linkage of Man3 were adjusted to φH = -27°, ψH = 35° to achieve agreement with the experimental NOE data.24 H-C-O-H dihedral angles (θHCOH) were varied in 30° increments and the corresponding distances were compared to the experimentally determined data by calculating root-mean-square deviations (RMSD). The lowest value of RMSD was considered to correspond to the optimum dihedral angle.

ASSOCIATED CONTENT Supporting Information. Complete lists of NOE-derived distances involving Man3 OH protons (Table S1), H6 protons (Table S2), and OH and NH protons (Table S3), parameter settings for all NMR experiments (Table S4), plot of the temperature dependence of OH protons (Figure S1), plots of NOE build-up curves (Figure S2), plots of OH proton distances vs θHCOH (Figure S3), plot of backbone carbonyl carbon chemical shift differences (Figure S4), plot of OH temperature coefficients vs hydrogen bond distances (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was supported by The Carl Trygger Foundation (G.N.), a Science Foundation Ireland Grant 13/IA/1959 (S.O.), and a National Institutes of Health Grant RO1GM080642 (A.M.G.). We thank Mike Delk for NMR technical support and Dr. Elena Matei for helpful discussions.

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Journal of the American Chemical Society

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Direct Observation of Carbohydrate Hydroxyl Protons in Hydrogen

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