(Received for publication, July 26, 1995; and in revised form, October 25, 1995)
From the
Linkages between sugar residues in branched oligosaccharides exhibit various degrees of flexibility. This flexibility, together with other forces, determines the overall solution conformation of oligosaccharides. We used the method of time-resolved resonance energy transfer to study the solution conformations of a biantennary glycopeptide and its partially trimmed products by exoglycosidases. The N-terminal of the glycopeptide was labeled with 2-naphthyl acetic acid as a fluorescent donor. Either terminal sugar residue, Gal6`, on the branch bearing 6-linked Man (antenna 6`), or Neu5Ac on the branch bearing 3-linked Man (antenna 6) was labeled with 5-dimethylaminonaphthalene-1-sulfonyl as an acceptor. The distance and distance distributions between the terminals were measured. In the intact biantennary glycopeptide, the donor-acceptor distance distribution of antenna 6` is bimodal with a majority of the population in the extended conformation and that of antenna 6 in one very broad population. The Neu5Ac on antenna 6 is oriented toward the N-terminal at low temperature and adopts a more extended form at high temperature. The removal of individual sugar residues along one of the two antenna in the biantennary oligosaccharide has a small effect on the distance distribution of the remaining antenna for both antennae 6 and 6`. Together with previous studies of the triantennary glycopeptides (Rice, K. G., Wu, P. G., Brand, L., and Lee, Y. C.(1993) Biochemistry 32, 7264-7270), our results suggest that both steric hindrance and inter-residue hydrogen bonding are very important in the folding pattern in oligosaccharide structures.
Conformational flexibility of linkages between sugar residues is an integral part of oligosaccharide structure. This is demonstrated in x-ray structures as multiple conformations (2) or conformations selected according to protein-carbohydrate interactions(3, 4, 5) . Oligosaccharides in solution are subject to thermal fluctuations and consequently they sample various conformations, which leads to heterogeneity in the structure. Great efforts both in the experimental and theoretical areas have been devoted to the determination of solution structure and conformation of oligosaccharides(6, 7, 8) .
In previous work, we used the method of time-resolved resonance energy transfer to determine the solution conformations of complex triantennary glycopeptides. Both extended and folded conformations of carbohydrate chains were observed (9) and the conversion between the two forms was temperature-dependent(10) . These results suggested that some linkages between sugar residues can rotate to a large extent under certain conditions. When the triantennary glycopeptides were treated with exoglycosidases to produce single chain isomers, the extended conformation was the dominant form(1) . Thus the intrinsic flexibility of linkages themselves does not automatically lead to a large conformational heterogeneity in oligosaccharides with multiple antennae. Other factors such as steric hindrance and hydrogen bonding may play roles in the solution behaviors of oligosaccharides.
In this work we present results on a biantennary glycopeptide and its partially digested products by exoglycosidases. We used time-resolved resonance energy transfer methods to determine the average end-to-end distances and distance distributions between the N-terminal and each one of the sugar terminals. Our aim is 3-fold. (i) How does the intact biantennary behave in solution compared to the triantennary glycopeptides reported recently? (ii) How does the sugar residue Neu5Ac behave at the end of the carbohydrate chain? (iii) How do the properties of the biantennary glycopeptides change when one or more sugar residues on one antenna are removed?
Time-resolved
fluorescence decays were measured on a photon-counting instrument with
a picosecond dye laser system as described earlier (10) . The
overall instrument response is about 60 ps. The excitation wavelength
was set at 290 nm and the emission wavelength of donor naphthyl was set
at 340 nm with a polarizer oriented at the magic angle 54.7°. A
dilute Ludox (silica) scattering solution was used to collect the
instrument response. The temperature of the cuvette was controlled by a
circulating water bath. Duplicate or triplicate data sets were
collected. The decay data were analyzed by nonlinear least squares as
described(9, 10) . Briefly, the donor decay in the
absence of acceptor dansyl ()was analyzed by a sum of
exponentials:
where and
are the amplitude
and lifetime of component i. The quality of fit was judged by the
reduced-
, weighted residuals, and the autocorrelation
of the residuals. A good fit was accepted when the reduced-
was between 1.0 and 1.2 with random residuals and autocorrelation
of the residuals. The average lifetime was calculated from =
/
.
The donor decay in the presence of dansyl was also analyzed empirically
by to obtain the average lifetime. Once the average
lifetimes of the donor in the absence and presence of the acceptor were
obtained, the energy transfer efficiency was calculated by E = 1 -
/
. The
average distance between donor naphthyl at the N-terminal and acceptor
dansyl at the antenna terminal was calculated according to = R
(1/E - 1)
, where R
is the Förster distance (22
Å at 20 °C) of the donor-acceptor pair evaluated as
before(9) . The donor decay in the presence of the acceptor was
analyzed by a model of distance
distributions,
where the first sum refers to the number of populations, each
with a concentration of a and a distance
distribution where R
is p
(r). The distance distribution was
modeled either as a Gaussian,
where is the average distance and is the standard
deviation. When the distance distribution is asymmetric, an asymmetry
parameter was used as before(12) . was used to fit
experimental data by nonlinear least squares, with the average
distance, the standard deviation, and the concentration as the
adjustable parameters.
Our previous results showed that there is a large fraction of folded conformation in both antennae 6 and 6` in the triantennary structure(9, 10) . When two antennae were completely removed (resulting in three single chain isomers), there is a substantial increase in the average donor-acceptor distance in antennae 6 and 6`(1) . Starting with a biantennary structure in this work, one might expect either that there has already been a large conformational change from a triantennary to a biantennary structure or that significant changes occur only from a biantennary to a single chain structure. The following results show that conformational changes are relatively minor from a biantennary to a single chain structure, thus implying that a substantial conformational change may have already occurred from a triantennary to a biantennary structure.
Figure 1: A, the structure of biantennary glycopeptide. B, the structure of triantennary glycopeptide.
(a) With a stepwise removal of sugars in either antenna 6 or 6`, the change in average distances is small from the biantennary to the single chain structures and is not uniform (or monotonic) in that the removal of either GlcNAc 5 or 5` leads to a minor decrease. Thus we do not expect a large shift in conformation from a biantennary to a single chain isomer.
(b) For antenna 6`, the average distance changes are 1.7 Å in the biantennary and 1.6 Å in the single chain structure from 1 to 40 °C. The decrease in distance is comparable with that of a similar single chain glycopeptide containing only a variation in the amino acids(1) , but not to that of antenna 6` in the triantennary(1, 10) . This single chain isomer serves as a reference when we compare our present results with previous ones. The average distance in the single chain antenna 6` is 17.5 Å at 20 °C, as compared to that of the similar single chain 6` 18.3 Å with a different amino acid substitution(1) . These distances are very close. The small difference in the average distances may be due to two sources: sample variations may account for about 0.2-0.3 Å and the difference in the interactions of naphthyl with the N-terminal amino acids may account for the rest (the average lifetime of the donor is about 36 ns here and compared to about 26 ns in the earlier sample, and the interaction is primarily from amino acids since the donor decay is nearly single exponential with 98% of the amplitude, excluding the possibility of interactions from folded-back sugar chains).
(c) In antenna 6, the average distance is 17.2 Å in the biantennary glycopeptide at 20 °C. This is about 3 Å shorter than that of the same antenna without Neu5Ac in the triantennary and about 6 Å shorter than that of the single chain isomer 6 lacking Neu5Ac(1) . Since there is one more sugar unit in antenna 6 of the biantennary structure, the end to end distance should be longer were all sugars in straight conformation. The shortening in the distance in this antenna implies that the linkage between Neu5Ac and Gal6 must be oriented such that Neu5Ac folds back toward the N-terminal in order to reduce the effective end to end distance. This is corroborated by the temperature dependence of the average distances. The change in the average distances from 1 to 40 °C in antenna 6 of the biantennary and single chain is about 0.5 Å, which is much smaller than that of antenna 6`. As shown earlier(1, 10) , the smaller change is due to some conformational change. In this case it can only occur between Neu5Ac and Gal6.
Figure 2: Distance distributions (peak-normalized) of antenna 6 in the intact biantennary glycopeptide as a function of temperature. One asymmetric distance distribution was used in the fit.
Figure 3: Distance distributions of antenna 6` in the intact biantennary glycopeptide as a function of temperature. Two distance distributions were used in the fit. The distributions are plotted in two ways: in A, the population of each distribution is proportional to the area; in B, is proportional to the peak value.
Fig. 4shows the change in distance distribution of antenna 6 when sugar residues Gal6`, GlcNAc5`, and Man4` on antenna 6` are sequentially removed. The removal of Gal6` has no noticeable effect on the distance distribution on antenna 6 and the cleavage of GlcNAc5` slightly broadens the distribution. Cutting Man4` leads to a small decrease in the width of the distribution. Nonetheless the distance distribution of the single chain 6 is quite broad.
Figure 4: Distance distributions (peak-normalized) of antenna 6 in the partially digested biantennary glycopeptides as a function of sugar residues removed from antenna 6`. Solution temperature was at 20 °C. Sugar residues trimmed are indicated in the figure.
Fig. 5shows the shift in the distance distribution of antenna 6` with sequential cleavage of sugar residues Neu5Ac as well as Gal6, GlcNAc5, and Man4 on antenna 6. The change is not uniform. In the intact chain (Fig. 1), two distance distributions are required to fit the data. Once Neu5Ac and Gal6 are removed, the decay can be fit with one asymmetric Lorentzian distance distribution (a similar fit to the intact chain leads to systematic deviations). However, when GlcNAc5 is cut, two populations are again required for the fit. Once all the sugar residues on antenna 6 are removed, the decay is fit with one asymmetric distance distribution. It should be noted that the difference between one asymmetric Lorentzian and distribution and two distance distributions is not as large as it appears in the figure. This is because the concentration of the folded form is about 25% at best, which is within borderline between one or two distance distributions when decay curves are analyzed. The real distance distribution may lie somewhere between the two forms of function, i.e. there is a small fraction of the folded form in antenna 6` and its concentration is slightly modulated by the removal of sugar residues in antenna 6. In the single chain 6`, the distance distribution is close to that of a similar single chain 6` reported recently(1) .
Figure 5: Distance distributions (peak-normalized) of antenna 6` in the partially digested biantennary glycopeptides as a function of sugar residues removed from antenna 6. Solution temperature was at 20 °C. Sugar residues trimmed are indicated in the figure.
The end to end distances of the biantennary glycopeptide and
those of its single chain isomers show some interesting features
compared to those of a triantennary glycopeptide and its single chain
isomers(9, 10) . In the triantennary glycopeptide, the
presence of antenna 8 apparently forces antennae 6 and 6` to fold back
half of the time(9) . In the biantennary glycopeptide, on the
other hand, the major portion of the population is in the extended
conformation in both antenna 6 and 6`. This implies that antenna 8 in
the triantennary glycopeptide has substantial steric effects on the
solution behaviors of other antennae. Thus steric hindrance is
important in determining the overall folding pattern of carbohydrate
chains. In antenna 8, all sugars except for Man4-Man3 are
1,4-linked, which are known to be conformationally rigid. The
rigidity of antenna 8, in tri-, bi-, and mono-antennary structures
suggests that
1,3-linkage in the Man4-Man3 is also a rigid one.
Therefore antenna 8 appears more rigid and forces other antennae to
adopt an alternative conformation in a crowded structure such as a
triantennary. A schematic illustration of the conformational
flexibilities in triantennary and biantennary glycopeptides is shown in Fig. 6. In the triantennary structure, there is an equilibrium
between the fold-back and open conformation in either antenna 6 or
antenna 6`, while in the biantennary structure, the equilibrium is
predominantly shifted toward the open form.
Figure 6:
An
illustration of conformational equilibria of different antennae in the
triantennary (A) and (B), and in the biantennary (C) and (D) glycopeptides. Antenna 6` can fold back
due to the flexibility in linkage 1-6 and/or
1-2,
while antenna 6 can fold back due to the mobile linkage
1-2. Square, N-acetylglucosamine; diamond, sialic
acid; triangle, galactose; and circle,
mannose.
Our results show that
Neu5Ac has orientations more toward the N-terminal. This can be viewed
as folded-back conformations as shown in Fig. 7. The
2-6-linkage, like
1-6-linkage(14, 15) , is flexible arising
from the rotation of several single bonds. Since this sugar residue is
at the end of the antenna, it is unlikely that steric hindrance is the
determining factor for the conformation observed. Another factor may be
hydrogen bonding between sugar residues. Molecular dynamics simulations
of the biantennary glycopeptide up to 200 ps have shown that Neu5Ac can
form many hydrogen bonds with various inner sugar
residues(13) . If a sufficient number of hydrogen bonds are
formed, a folded-back conformation can be stabilized. As solution
temperature increases, more motions are produced, which leads to
breakage of hydrogen bonds. This in turn results in a more extended
conformation for Neu5Ac. The shift to an extended conformation
compensates the temperature-enhanced intramolecular diffusion between
the two ends (which reduces apparent distances when this motion is not
included in data analysis) so that a smaller apparent change in average
distances is detected.
Figure 7: A schematic presentation of the equilibrium between an open or extended form and a fold-back form of sialic acid (SA or Neu5Ac) in antenna 6 of the biantennary glycopeptide (Fig. 1).
The wide distance distribution of the single
chain 6 can be understood in the following. From the results of Rice et al.(1) , the single chain 6 lacking Neu5Ac has
quite a wide distance distribution. This can be attributed to the
flexibility of 1,2-linkage between GlcNAc5 and Man4. The linkage
between Neu5Ac and Gal6 is the
2-6 type, which is also
expected to be flexible. The combination of the two factors are thus
responsible for the asymmetric distance distribution.