Department of Chemistry and Biochemistry, University of Maryland, 5401 Wilkens Avenue, Baltimore, MD 21228-5398
Email:
qiuxu@model.chem.umbc.edu or bush@model.chem.umbc.edu
Polysaccharides and glycoproteins on cell surface have been recognized to play many important roles in cell-cell recognition and protein folding. Within the cell, the N- linked carbohydrates of glycoproteins influence protein folding. Study of their chemical structures, conformations, and dynamics may in the future help us better understand their roles in biological systems and assist in designing drugs to block the lectin- carbohydrate interaction. The question of flexibility of oligosaccharide has been controversial (Homans, 1993; Carver, 1991,1993). Some workers have presented results which were interpreted as indicating that the stereochemical constraints of the glycosidic linkage are much more restrictive than those of the peptide bond and that oligosaccharides have much more restricted motion than peptides (Bush, 1992). Other research groups have described results which indicate that oligosaccharide motions are generally not very restricted and that oligosaccharide epitopes do not have a single discrete conformations. All these conclusions have been based on reconciliation of molecular dynamics simulations with NOE data, fluorescence energy transfer and some additional support from far ultraviolet circular dichroism spectra. However, interpretation of NOE data in systems with conformational fluctuation is difficult and some workers have claimed that it is possible to misinterpret NOE data for flexible oligosaccharides and to incorrectly conclude that they are rigid. As a result of uncertainty in the interpretation of NOESY data for flexible systems, it has been proposed that long range coupling constants (3JCH) might be a useful adjunct for testing the validity of rigid models and as an additional source of conformational data necessary to refine the parameters needed to accurately describe flexible models. We show that the antiphase methods for measuring 3JCH in oligosaccharides have limited reliability but that the coupling constants can be reliably measured in natural abundance by quantitative J-correlation methods, and in 13C uniform enrichment by E. COSY type methods. Here we present the results of conformational search on a rigid milk pentasaccharide, LNF2, a flexible cell wall oligosaccharide S. gordonii 38 , and a flexible cell wall polysaccharide of S. mitis J22 (Structures are shown in Table 1). The first two oligosaccharides are in natural abundance, and the last polymer is in 13C uniform enrichment.
The sample preparation of these three saccharides have been reported previously (Bush et al. 1985; Gitti et al. 1994; Xu et al. 1996a). All the NMR sample was dissolved in 99.96% D2O of 450 ml after several times of deuterium exchange. The concentration for LNF2 was about 60 mg/ml, for S. gordonii 38 was about 20 mg/ml, and for S. mitis J22 was about 10 mg/ml. The NMR spectra were acquired on either GE GN-500 or GE Omega-500 PSG spectrometer. The temperature was set at 7 C, 24 C, and 25 C for the samples S. gordonii 38, S. mitis J22 and LNF2 respectively. All the spectral parameters are listed in the figure legends. For natural abundance oligosaccharide, LNF2, the 3JCH was measured with SQMBC (Norwood et al. 1990) and quantitative 2D HMQC (Zhu and Bax 1993). The reference spectra for SQMBC were proton 1D and HOHAHA. The reference spectrum for QHMQC was obtained from the same pulse sequence except for the replacement of hard carbon pulses with delays of the same time. For the 13C enriched polysaccharide, S. mitis J22, the 3JCH was measured from 13C-1H coupled 2D NOESY and 3D HMQC-NOESY according to E. COSY type spectra. A 2D NOESY spectrum was run on S. gordonii 38, and a 3D 13C decoupled HMQC-NOESY was acquired on 13C enriched S. mitis J22 polysaccharide. The procedures of molecular modeling of the polysaccharide of S. mitis J22 based on NMR 3JCH and NOE data are in preparation and will be presented elsewhere.
| Table 1 Chemical Structures of Oligosaccharides and Polysaccharides |
|
Lacto-N-fucopentaose 2 (LNF2):
betaGalp(1->3)-betaGlcpNAc(1->3)-betaGalp(1->4)-alfabetaGlc
|
alfaFuc(1->4)
|
|
Oligosaccharide from S. gordonii 38:
alfaGalpNAc(1->3)-betaRhap(1->4)-betaGlcp(1->6)-betaGalf(1->6)-betaGalpNAc(1->3)-alfabetaGalp
|
alfaRhap(1->2)
|
|
Repeating subunit of polysaccharide from S. mitis J22 :
a b c d e f
6)alfaGalpNAc(1->3)-betaRhap(1->4)-betaGlcp(1->6)-betaGalf(1->6)-betaGalp(1->3)-alfaGalpNAc(1->PO4->
|
alfaRhap(1->2)
g
|
A Rigid Oligosaccharide
From the SQMBC spectrum, the nJCH were extracted from the antiphase splitting by comparison ("fitting") of the line shape of a f2 cross section through a given long range correlation with a corresponding reference line shape from either 1D proton or HOHAHA spectrum (Figure 1a and 1b). The long range coupling constants in the range of 3.3-6.6 Hz for LNF2 can be extracted quite accurately from this antiphase method. However, our results for broader proton multiplets were less satisfactory. We conclude that this method will not give reliable values for small coupling constants. As a result of the limited reliability of SQMBC method for this pentasaccharide, we have applied the QHMQC method. The long range coupling constants of LNF2 were calculated from the ratio of correlation and reference peaks. In contrast to the SQMBC method, for which only curve shape can be fitted, both the shape and magnitude of QHMQC were used in the spectral fitting.
The reliability of nJCH measurement by QHMQC over SQMBC is reflected in Table 2 and Figures 2 and 3, which show good agreement between our values and those by Morat et al. (1988) for substituted monosaccharides. The values of 3JCH across the glycosidic linkages in LNF2 are presented in Table 3 together with the calculated values from a rigid model by Cagas and Bush (1990). The standard deviation by this method, we estimated, is ±0.2 Hz. Since the error is dictated by the signal to noise ratio in QHMQC, it is the same absolute value for large or small coupling constants. Comparing the data of our QHMQC and those of SQMBC by Kogelberg and Rutherford (1994), all agree well except for a small 3JH1gal-C4glcNAc of 2.7 Hz. Our value is smaller than that by Kogelberg and Rutherford. The error range of SQMBC was reported larger than the range of QHMQC. We propose that the antiphase method of SQMBC gives an over-estimate for a small coupling constant due to antiphase cancellation.
The method used for calculation of the coupling constants in Table 3 (Cagas and Bush, 1990), is essentially identical to that derived from molecular dynamics simulations by Mukhopadhyay and Bush (1990) and is similar to the model of Kogelberg and Rutherford (1994). We conclude that the measured values of 3JCH for LNF2 are in good agreement with a rigid model predicted by molecular modeling calculations and with NOE data.
A Flexible Oligosaccharide
The 3JCH across the glycosidic linkages of cell surface polysaccharide of S. mitis J22 was measured from 2D NOESY and 3D HMQC-NOESY without 13C decoupling. The data are listed in Table 4. The molecular modeling on the first four residues, which constitute an antigenic site, has been carried out (Xu et al. 1996a). The result shows that no single conformer can reproduce the experimental 3JCH, an ensemble of conformation with different statistical weights are necessary to match the experimental results. The conformation of the heptasaccharide has been searched based on linear matching of 3JCH between experiment and model with the program based on the singular value decomposition (SVD) (Press et al. 1992, Xu and Bush. 1996c). The statistical weights of the multiple conformers and the calculated 3JCH are shown in Table 5 and 4. With the same statistical weights from the 3JCH match, proton NOE intensities were simulated according to the complete relaxation matrix method (Keepers and James, 1984; Bush 1994) and compared to the experimental data from 3D HMQC-NOESY (Table 6). We observed that 1H-13C dipolar relaxation is an important pathway in NOE development in this 13C enriched sample, and this hetero-relaxation rate is included in auto-relaxation rates. The effective rotational correlation time (taue=1.4 ns) was adjusted to find a best fit between experimental and simulated data on intra-residue 1H-1H NOE. The structures of the first four conformers in Table 5 are shown in Figure 4.
Similar procedures were taken to simulate NOE for antigenic conformation ofS. gordonii 38 at natural abundance at 7 C. A good match between experiment and simulation was observed with an effective rotational correlation time of 1.8 ns (Xu, et al. 1996b, data not shown here). The conformers and statistical weights to simulate NOE for the antigenic site were those from the result by Xu et al. (1996a).
Using 3JCH across the glycosidic linkages to define oligo, polysaccharide or glycoprotein conformation is attractive since the degree of freedom defined by the dihedral angles is directly related to conformation, and the data interpretation is free from the assumption of motional dynamics of the molecule.
In determining long range hetero-nuclear coupling constants for a natural abundance sample, the antiphase methods like SQMBC suffer antiphase cancellation for small values. QHMQC gives data with low error range even for relatively small coupling constants. The primary limitation for the latter is the requirement for an isolated proton multiplet for use as a reference, which has been appropriately overcome with one bond HMQC spectrum scaled to obtain a reference spectrum (Zhu et al. 1994). A second limitation, which is not so easy to overcome, is that a very large oligosaccharide sample is needed. The signal-to-noise ratio for the QHMQC can be considerably degraded by the two long delays in the pulse sequence.
The location of multiple conformers by the 3JCH data for the S. mitis J22 polysaccharide is confirmed with the match between the simulated and experimental NOE. The quality of NOE match is judged by crystallographic-type R-factor and sixth- root weighted Rx-factor (James, 1991). R=0.01 and Rx=0.002 for the 13C enriched polysaccharide (Table 6). The excellent match of experiment and simulation for both 3JCH and NOE for this polysaccharide compared to similar fits for DNA samples indicates that conformational search according to 3JCH across the glycosidic linkages is a better choice.
It is quite clear that the blood group oligosaccharide LNF2 is rigid, S. mitis J22 polysaccharide and S. gordonii 38 oligosaccharide are flexible. In addition, there is variation in flexibility within the latter molecules. There are two lines of evidence: firstly the dihedral angles across the glycosidic linkages and P(Galf) show that the range and the extent of variation are different along the heptasaccharide (Table 7); secondly dynamics data (Xu & Bush, 1996d) show that the order parameters, which reflect the magnitudes of motion, vary from residue to residue (Figure 5). The residue d (Galf) is more flexible than the residue b (Rha). We have found there are at least two time scale motions (pico-second and nano-second) which superimpose on the overall tumbling of this polymer. The variation among the residues for pico-second motion is much smaller than that for nano-second motion. This could imply that pico-second motion (ring puckering) is common, while the nano-second motion (dihedral angle hinging) depends on local spatial constraints. Further experiments are necessary to check the population and time scale of pico and nano second motions in both rigid and flexible polysaccharides.
| Conclusion |
1. 3JCH is a reliable experimental data to search for conformation, especially flexible polymer or oligomer.
2. For natural abundance sample, antiphase method (SQMBC) is limited to large nJCH, however, QHMQC can measure 3JCH small with little error.
3. Saccharide or glycoprotein can be rigid and flexible depending on their primary and secondary chemical structures.
| Table 2 Intraresidue nJCH for the pentasaccharide, Lacto-N-fucopentaose 2, determined by quantitative coherence transfer |
| nJCH | nJCH (expt.1) | nJCH (lit.2) | |||
| b-Gal-3 | H2-C2-C1 | 2JC1/C2 | 6.1 | 6.3 | |
| H2-C2-C3 | 2JC3/H4 | 6.5 | 6.8 | ||
| a-Fuc | H5-C5-C4 | 2JC4/H5 | 4.23 | 5.2 | |
| H1-C1-C2-C3 | 3JC3/H1 | 5.3 | 5.1 | ||
| H1-C1-O-C5 | 3JC5/H1 | 6.5 | 6.7 | ||
| b-GlcNAc | H4-C4-C3 | 2JC3/H4 | 4.1 | 4.2 | |
| b-Gal-4 | H4-C4-C3 | 2JC3/H4 | 4.5 | 4.7 | |
| H4-C4-C3-C2 | 3JC2/H4 | 5.1 | 5.8 | ||
| a-Glc | H1-C1-C2-C3 | 3JC3/H1 | 5.7 | 5.6 | |
| H1-C1-O-C5 | 3JC5/H1 | 6.5 | 6.9 |
1. Values in Hz 2. Values reported by Morat et al. (1988) 3. t1 noise present in this region
Table 3 Interglycosidic coupling constants of the blood group oligosaccharide LNF2 in D2O 3JCH Cal.(1) Exp. H3GlcNAc-C3GlcNAc-O-C1Gal3 (phi) 3JC1/H3 5.0 5.7 H1Gal3-C1Gal3-O-C3GlcNAc (psi) 3JC3/H1 3.4 2.7 H4GlcNAc-C4GlcNAc-O-C1Fuc (phi) 3JC1/H4 5.0 H1Fuc-C1Fuc-O-C4GlcNAc (psi) 3JC4/H1 2.5 4.0 H1GlcNAc-C1GlcNAc-O-C3Gal4 3JC3/H1 3.1 H1Gal4-C1Gal4-O-C4abGlc 3JC4/H1 3.2 1. Calculated values from conformer 3 of Cagas and Bush (1990)
Table 4 Experimental and calculated 3JCH of polysaccharide S. mitis J22 Cal.(a) Exp. Cal. Exp. 3JCH(phiH)ab 1.70 1.50 3JCH(psiH)ab 2.31 1.50 3JCH(phiH)bc 1.73 2.00 3JCH(psiH)bc 2.10 2.10 3JCH(phiH)gb 1.48 1.50 3JCH(psiH)gb 3.39 4.10 3JCH(phiH)cd 1.46 1.50 3JCH(psiH)cd 2.18 1.90 3JCH(phiH)de 2.04 1.50 3JCH(psiH)de 1.40 1.30 3JCH(phiH)ef 2.71 2.40 3JCH(psiH)ef 2.36 1.30 sigma=0.50 (Hz) a. 3JCH calculated from statistical weights of Table 5 and the dihedral angle correlation of Tvavoska et al. (1989)
Table 5 Statistical weights of conformers from linear 3JCH fitting Conformer coefficient pi conformer 322-7 0.4369 conformer 322-48 0.3247 conformer 422-82 0.2343 conformer 422-239 0.0009 conformer 822-71 0.0013 conformer 822-182 0.0019
Table 6 13C enriched polysaccharide of J22 with 350 ms mixing at 24.0 C Cross Cal. Exp. Cross Cal. Exp. -Peaks -Peak aH1-bH1 0.0053 0.0052 bH5-cH2 0.0007 0.0070 aH1-bH2 0.0488 0.0340 gH2-cH2 0.0001 0.0003 aH1-bH3 0.0237 0.0159 gH2-cH5 0.0004 0.0019 aH1-bH4 0.0030 0.0051 gH3-cH5 0.0042 0.0004 aH1-bH5 0.0019 0.0010 gH5-cH5 0.0169 0.0081 aH1-gH1 0.0285 0.0130 cH1-dH6 0.0301 0.0313 aH1-gH4 0.0007 0.0008 cH1-dH6'0.0240 0.0226 aH1-gH5 0.0040 0.0017 cH1-dH5 0.0040 0.0143 aH2-bH1 0.0006 0.0005 cH1-dH4 0.0046 0.0069 aH2-bH2 0.0050 0.0062 cH2-gH2 0.0001 0.0003 aH2-bH3 0.0031 0.0018 cH2-dH6 0.0019 0.0015 aH2-gH1 0.0050 0.0038 cH2-dH6'0.0020 0.0023 aH2-gH5 0.0005 0.0020 cH2-dH5 0.0006 0.0022 aH3-bH3 0.0032 0.0037 cH2-dH4 0.0018 0.0020 aH3-bH1 0.0019 0.0011 cH2-eH3 0.0001 0.0037 aH3-gH1 0.0144 0.0063 dH5-fH5 0.0001 0.0010 aH4-bH3 0.0033 0.0051 dH4-dH1 0.0038 0.0026 aH5-bH1 0.0035 0.0023 dH4-eH5 0.0013 0.0006 aH5-bH3 0.0374 0.0256 dH4-fH5 0.0012 0.0094 aH5-bH4 0.0127 0.0033 dH1-dH2 0.0289 0.0147 aH5-bH5 0.0032 0.0014 dH1-dH3 0.0035 0.0057 aH5-gH1 0.0081 0.0038 dH5-dH1 0.0016 0.0017 aH6-bH3 0.0025 0.0054 dH1-eH1 0.0005 0.0003 aH6'-bH30.0038 0.0045 dH1-eH4 0.0019 0.0035 bH1-cH1 0.0013 0.0019 dH1-eH5 0.0037 0.0035 bH1-cH2 0.0025 0.0079 dH1-eH6 0.0241 0.0129 bH1-cH3 0.0130 0.0197 dH1-eH6'0.0269 0.0099 bH1-cH4 0.0196 0.0437 dH1-fH4 0.0012 0.0031 bH1-cH5 0.0026 0.0040 dH2-eH4 0.0030 0.0038 bH1-gH1 0.0020 0.0084 dH2-fH2 0.0001 0.0002 bH1-gH5 0.0039 0.0053 dH2-fH4 0.0006 0.0010 bH2-gH1 0.0174 0.0253 dH2-fH5 0.0001 0.0043 bH2-gH5 0.0283 0.0120 eH1-fH2 0.0025 0.0077 bH2-cH1 0.0013 0.0004 eH1-fH3 0.0501 0.0282 bH2-cH3 0.0164 0.0043 eH1-fH4 0.0194 0.0058 bH2-cH4 0.0029 0.0066 eH1-fH5 0.0041 0.0018 bH2-cH5 0.0025 0.0027 eH2-fH3 0.0025 0.0017 bH3-gH1 0.0049 0.0029 eH4-fH3 0.0008 0.0083 bH4-gH1 0.0118 0.0034 eH5-fH3 0.0047 0.0084 bH4-gH2 0.0074 0.0034 eH5-fH4 0.0081 0.0012 bH4-cH2 0.0002 0.0011 eH5-fH2 0.0004 0.0007 R = 0.01 Rx = 0.002 a. NOE are calculated from the statistical weights of Table 5
Table 7 Glycosidic dihedral angles of conformers of flexible polysaccharide from S. mitis J22 a-b b-c g-b c-d d-e e-f phi psi phi psi phi psi phi psi gamma omega Phase phi psi omega phi psi Conf. 322-7 52.2 71.1 44.9 67.6 -54.8 159.0 -64.7 -176.0 -87.0 65.5 -27.3 -29.7 180.0 -165.0 -68.2 -169.0 Conf. 322-48 52.9 66.3 49.2 69.8 -59.5 162.0 -49.1 98.5 -81.2 35.8 37.8 -169.0 -174.0 37.3 -80.2 175.0 Conf. 422-82 82.6 168.0 140.0 62.6 -66.7 154.0 -57.8 -160.0 -79.5 68.0 -42.4 -151.0 -176.0 68.0 -77.8 -158.0 Conf. 422-239 79.3 160.0 135.0 63.2 -83.4 143.0 -31.2 -65.5 -67.9 172.0 -28.0 -65.2 -93.3 60.9 -85.5 -148.0 Conf. 822-71 63.1 104.0 88.3 -46.5 -85.8 77.0 -67.3 -139.0 -78.6 65.3 -44.9 -47.6 172.0 170.0 -76.3 -166.0 Conf. 822-182 61.3 108.0 59.2 -78.4 -86.2 122.0 -3.48 75.6 -79.3 160.0 -29.0 -62.4 -81.3 75.9 -33.8 -149.0
| References |
| Figure legends |
Figure 1: Examples of long range coupling constant measurement by the SQMBC method for LNF2. a ) 3JC5- H1 of a-Glc. Upper (dotted) curve shows f2 cross section from the SQMBC spectrum and lower (solid) curve shows best fit of the reference spectrum and three lower curves show three equally good fits of the reference spectrum.
The experiments were recorded at 25 C with a spectral width of 2.27 KHz in f2. Two sets of 172x2K complex data matrices were acquired in alternating blocks with 256 scans per t1 and digital resolution of 1.1 Hz/point in f2, and 41.5 Hz/point in f1. Zero filling in f1 resulted in a 2Kx2K real data matrix. Reference multiplets were extracted from a 1D proton spectrum or from a 2D HOHAHA spectrum recorded with the pulse sequence of Bax and Davis (1985) with a mixing time of 70 msec. The spectrum was recorded as a 2x384x2K data matrix with digital resolution of 1.1 Hz/point in f2 and 5.9 Hz/point in f1. Zero filling in f1 resulted in 2Kx2K real spectrum.
Figure 2: Selected examples of f2 cross sections (solid lines) and reference line shapes (dotted lines) used to calculate some interglycosidic nJCH of LNF2 by the QHMQC method.(Zhu and Bax, 1993). Scale factors used in fitting are given in the figure.
The quantitative 2D HMQC spectra were acquired using the pulse sequence of Zhu and Bax (1993): Modified phase cycling was used for phase sensitive acquisition (States et al., 1982). The 2D "reference spectrum" was obtained as reported originally (Zhu and Bax,1993), with 90¡ 13C pulses replaced by delays of 34 msec which was the 90¡ 13C pulse width used in this experiment. Both spectra were recorded at 24 C with D = 40 msec using 83 msec dwell time in the t1 dimension and 208 t1 increments, with 96 scans per t1 increment for the 1H-13C spectrum and 8 scans for the reference spectrum. Two sets of 208 x 1024 complex data matrices were collected in alternate blocks for the 1H-13C correlation spectrum while for the 2D reference spectrum one 208 (real) x 1024 (complex) matrix was recorded and zeros were inserted for each t1 value of the imaginary data prior to the t1 Fourier transformation as described by Zhu and Bax (1993). Cosine-bell apodization was used in both t1 and t2 dimensions and the final zero-filling resulted in digital resolution of 2.8 Hz/point and 12 Hz/point in f2 and f1 respectively. The FELIX program was used to process the HMQC data and to calculate the ratio of the intensities of the correlation and reference spectrum. In this study we applied the "scaling factor" approach, which was previously reported (Zhu and Bax, 1993) to give better sensitivity. In particular rows through the corresponding cross peaks were inverse Fourier transformed and zero-filled to 2K real points to provide a resolution of 1.4 Hz/point in f2. Following the back Fourier transformation the corresponding correlation peak was multiplied by a factor which gives "best fit" between the reference and the correlation peak.
Figure 3: Illustration of the precision of the HMQC method for measurement of a)3JC1(gal3)-H3(glcNAc) and b)3JC3(glcNAc)-H1(gal3) of LNF2 by using different scaling factors to get the "best fit" between f2 cross sections of the 1H-13C correlation (solid lines) and the corresponding reference line shapes (dotted lines).
Figure 4: Conformers 322-7, 322-48, and 422-82 of repeating units in polysaccharide S. mitis J22
Figure 5: Order parameters were from fitting 13C relaxation rates and NOE with a "model-free" formalism by Clore et al. (1990). The overall rotational time is 4.7 ns. The tf is on pico-second and ts is at one nano-second.
