J
give direct information on the glycosidic dihedral angles 
and
. Averaging in the presence of internal motion is straight forward.
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
J
over multiple conformations
makes it possible to construct
a flexible model for the receptor
polysaccharide of Streptococcus mitis J22 from long range
heteronuclear coupling constants. Accurate coupling constants
were derived from 3-d HMQC NOESY data on a unformly 
C enriched
polysaccharide without decoupling.
Statistical weights of different conformers were extracted from linear fitting
to the experimental data to
J
values calculated from a Karplus relation.
The NOE data simulated from a statistically averaged complete
relaxation matrix were also in agreement with experimental NOE data
from a 
C decoupled 3-d HMQC-NOESY spectrum. The dynamic behavior
of the the 
C labeled polysaccharide was studied by T
,
T1rho and heteronuclear NOE measured with inverse detection.
Selective excitation of the anomeric carbon atoms
was used in order to simplify
data analysis. The relaxation data were fit to various dynamic models
of internal mobility for comparison with the structural models to provide
insight into the role of polysaccharide dynamics in
coaggregation of oral bacteria.
While models for rigid oligosaccharides can be derived from NOE
data and molecular modeling,
flexible oligosaccharide models require more extensive experimental data.
Interpretation of NOESY data in the presence of internal molecular
motion is difficult. But heteronuclear long-range coupling constants J
give direct information on the glycosidic dihedral angles and
. Averaging in the presence of internal motion is straight forward.
We report NOESY data and 
J
data for the heptasaccharide repeating
unit of the cell wall polysaccharide from Streptococcus mitis
J22 which is a lectin receptor for coaggregation of oral bacteria.
Structure of S. mitis J22 polysaccharide
Uniformly 95% 
C labeled
polysaccharides were obtained by growing Streptococcus mitis strain
J22 in 
C labeled media (Gitti et al., 1994).
Heteronuclear three-bond coupling constants, 
J
, across the glycosidic
linkage can be accurately measured from offsets of peaks in three-dimensional
HMQC-NOESY experiments without decoupling. (See Figure 1) In contrast
to antiphase methods, this
method gives accurate values for small coupling constants even for high
molecular weight polymers. Measured values in Table 1.
Table 1 Experimental and calculated 3JCH of polysaccharide S. mitis J22
Cal.(HZ) Exp.(Hz) Cal.(Hz) Exp.(Hz) 3JCH(phi)ab 1.70 1.50 3JCH(psi)ab 2.31 1.50 3JCH(phi)bc 1.73 2.00 3JCH(psi)cb 2.10 2.10 3JCH(phi)gb 1.48 1.50 3JCH(psi)gb 3.39 4.10 3JCH(phi)cd 1.46 1.50 3JCH(psi)cd 2.18 1.90 3JCH(phi)de 2.04 1.50 3JCH(psi)de 1.40 1.30 3JCH(phi)ef 2.71 2.40 3JCH(psi)ef 2.36 1.30
C
Good resolution for accurate measurement of NOESY cross peaks is
obtained by decoupled three-dimensional HMQC-NOESY spectra of the
C enriched polysaccharide.
(See Figure 2 and Table 2.)
Table 2. 13C enriched polysaccharide of J22 with 350 ms mixing at 24.0 C
Peaks Cal. Exp. Peak Cal. Exp. 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 = 0.01b Rx = 0.002b a. NOE are calculated from the statistical weights of Table 4 with an effective correlation time of 1.4 ns b. R and Rx are crystallographic-type R-factor and sixth-root weighted Rx-factor (James, T. L. 1991)
Interpretation of Experimental Data by Molecular Modeling
Since dihedral angle mapping and restrained molecular dynamics was unsuccessful for
building flexible models, starting conformations were generated
from the 
J
data using glycosidic dihedral angles calculated from eq. 1.
(Tvaroska et al., 1989)
where 
or 
.
The glycosidic dihedral angle 
is defined by the four atoms,
H
-C
-O-Cx and 
is defined by the four atoms,
C
-O-Cx-Hx.
The dihedral angles following IUPAC 
, defined by O
-C
-O-Cx
and 
defined by C
-O-Cx-C
are simply related to
the above angles.
Since no single low energy conformation fits all the 
J
data or the NOESY data,
a linear combination of six conformations (Equation 2) was sought by singular value decomposition
which could fit the 
J
data.
In our model, six conformations (Table 3) are in
exchange with statistical weights given in Table 4. The model fits the 
J
data within experimental error (
0.5 Hz) and fits the NOESY data calculated
from a complete relaxation matrix method assuming exchange rapid compared to T
and slow compared to the effective rotational correlation time (
= 1.4 ns).
Table 3 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
Table 4 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
The model described in Table 4 and Figures 3-5,
Figure 6
shows the antigenic site of the
polysaccharide, residues a, b, c, and g is more rigid than the
lectin-binding site, residues d, e and f.
Fluctuations in the conformation of the
- linkages and in the puckering of the 
- galactofuranoside produce large changes
in the chain direction (Figures 3-5,
Figure 6).
J
values by:
The force constant K
of the constraint was set as 1.0 kcal/mol rad
.
The atomic force from the above penalty function (Equations 1 and 2) required for
molecular dynamics simulation is obtained from the partial derivatives:
where 
is the distance between C and H.
The restraints cause the trajectories to be
confined to much smaller area of conformational space. While the
trajectory started from conformation 322-7
shows reasonable agreement of calculated 
J
with experimental values (Table 5), it is not so good as the multiconformation model of Table 4.
Table 5 MD and r-MD time-averaged coupling constants (Hz)
MD | r-MD 322-7 322-48 422-82 | 322-7 322-48 422-84 3JCH(phi)ab 1.4 1.1 4.1 | 1.4 1.5 3.1 3JCH(psi)ab 1.9 2.0 2.3 | 1.7 2.2 2.2 3JCH(phi)bc 4.4 5.2 4.1 | 3.2 1.1 4.8 3JCH(psi)bc 3.8 3.2 3.6 | 2.9 2.5 11.9 3JCH(phi)gb 2.8 1.7 4.2 | 1.0 1.8 2.5 3JCH(psi)gb 3.6 2.8 4.6 | 3.2 3.9 4.0 3JCH(phi)cd 2.7 0.7 2.5 | 2.3 1.1 1.6 3JCH(psi)cd 2.5 3.9 2.1 | 0.7 3.9 0.5 3JCH(phi)de 1.5 1.9 1.0 | 0.6 2.1 2.9 3JCH(psi)de 3.2 4.7 2.1 | 1.8 0.9 1.4 3JCH(phi)ef 2.2 2.9 3.1 | 2.6 1.9 2.4 3JCH(psi)ef 3.6 3.2 2.7 | 2.2 3.2 2.6 sigma 1.3 1.7 1.5 | 0.77 0.94 1.2 R 0.003 0.05 0.20 | 0.26 0.06 0.34 Rx 0.0007 0.012 0.053 | 0.053 0.014 0.099
C relaxation rates for anomeric carbon atoms of
the fully labeled polysaccharide were measured by indirect 
H
detection using the pulse sequences of Kay and coworkers, (Yamazaki et al., 1994) without
gradients (Figure 9).
The use of 
C pulses selective for the anomeric carbon atoms eliminates a number
of potential artifacts in measurement of T
, T1rho and heteronuclear NOE.
The decay rates in Table 6 are single exponential and are independent of 
C
carrier position (Figure 10).
T
and heteronuclear NOE are similar for the seven residues, but T1rho
values for the anomeric carbon atoms of the lectin binding site ( d, e and f) are significantly longer
indicating a different type of motion. (Figure 11)
Since little is known of the detailed dynamics of complex carbohydrates, any
model for internal motion
is subject to many uncertainties. A simple Lipari-Szabo model with a
overall tumbling modulated by a fast internal motion does not fit the data well.
An extension of this model (Clore et al., 1990) in which a slow internal motion
(nanosecond) is added to the rapid internal motion (picoseconds) (Equation 5) fits the data
well with an
overall tumbling time of 4.7 ns, (Table 6). The order parameters for
the fast (picosecond) internal motion are similar for all residues, while the
order parameter (Figure 12) for the slower internal motion is lower for residues d and
e emphasizing the greater conformational flexibility of the lectin binding
site in agreement with the model building based on 
J
(Table 4 and Fig 3 to 6).
where tauR'=tauR taui/(tauR + taui), i=f,s, and S2=Sf2 Ss2
Table 11 Experimental data of 13C relaxation rates
residue R1 ± 0.07 s-1 R1r ± 0.12s-1 NOE ± 0.13 R1r/R1 a 1.22 10.33 1.08 8.47 b 1.28 11.43 1.05 8.93 g 1.32 10.75 1.03 8.14 c 1.11 7.97 1.26 7.18 d 1.10 5.26 1.20 4.78 e 1.19 7.06 1.14 5.93 f 1.16 9.17 1.19 7.90
We interpret the picosecond motion as sugar ring puckering and the nanosecond motion as exchange between conformations of the type shown in Figures 3 to 6 which change mainly the conformational hinge of the lectin-binding site. Slower internal motions are responsible for the effective correlation time which describes motion of a larger region of the polymer.
Although anisotropy of the motion could contribute, similarity of
NOE data on an isolated
heptasaccharide subunit from this system argues against this possibility
(Xu et al., 1996b) as does
the similarity of the effective 
for the C1-H1 vectors of residues
a, b, c and g which point in quite different directions in the
models of Figures 3 to 6. The possibility of contributions to the T1rho
by conformational exchange is under study by measurements as a function of
static field B
and spin-locking field B
.
Clore, G. M., Szabo, A., Bax, A., Kaym L., Driscoll, P. C., and Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 4989-4991 Gitti, R., Long, G., Bush, C. A. (1994) Biopolymers 34, 1327-1338 James, T. L. (1991) Curr. Opin. Struct. Biol. 1, 1042-1053 Lipari, G., and Szabo, A. (1982) J. Am. Soc.104, 4546-4559 Press, W. H., Flannery, B. P., Teukolsky, S. A., and Vetterling, W. T. (1989) "Numereical Recipes", Cambridge University Press Tvaroska,I., Hricovini,M. and Petrakova,E. (1989) Carbohydr. Res. 189, 359-362. Xu, Q. W., Mohan, R., and Bush, C. A. (1996a) Biopolymers 38, 339-353 Xu, Q. W., Gitti, R., Bush, C. A. (1996b) Glycobiology 6, 281-288 Ymazaki, T., Muhandiram, R., and Kay, L. E. (1994) J. Am. Soc. 116, 8266-8278
