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HELICAL A segment of the carbohydrate coat
of group B Streptococcus, shown in two views, forms a helix
that antibodies can recognize.
COURTESY OF ROBERT WOODS |
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Nobody would argue with the statement that proteins are a challenge
for computer modelers. Descriptions of their swirls of helices
and ribbons, to say nothing of their further folding, can tax
even the biggest supercomputer. But the complexity of proteins
pales in comparison to that of their biochemical allies, the carbohydrates.
Though proteins may contain many thousands of atoms bunched
together in elaborate structures, they tend to hold their globular
shapes. Carbohydrates, by contrast, are bendy, twisty, frequently
branched strands that are often even bigger than proteins. And
that has made describing their behavior with computer simulations
a difficult prospect.
But there's a wealth of powerful information about carbohydrates
that can be gleaned from modeling, with profound implications
for biology and medicine, from infection to immunity. For example,
an intimate knowledge of the polysaccharide surface of a bacterium
could be key in pinpointing targets for antibiotics or vaccines.
And what scientists learn from studying carbohydrates could shed
light on other flexible biopolymers such as RNA and provide insights
into protein behavior as well.
Fifteen years ago, scientists' ability to model carbohydrates
lagged far behind their ability to model proteins and nucleic
acids. But carbohydrate chemists have been catching up, thanks
in part to giant strides made in computer power and speed. That
much was clear from a symposium on the computational chemistry
of carbohydrates that was held last month at the American Chemical
Society national meeting in Anaheim, Calif., and that was sponsored
by the Division of
Carbohydrate Chemistry.
"I would say, based on the symposium in Anaheim, that the gap
has been essentially closed," said John
W. Brady, chemistry professor in the food science department
at Cornell University. "The modeling being done now on carbohydrates
is as sophisticated as that being done on proteins."
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BEST BINDER Computer modeling helped select
the best binding conformation of wheat germ agglutinin and a
carbohydrate.
COURTESY OF GÖRAM WIDMALM |
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Nearly two dozen researchers from around the globe gathered to
discuss new advances in the field, from strategies for simulating
solvation to new computer programs tailored to predict carbohydrates'
unique characteristics. In particular, the coupling of nuclear
magnetic resonance experiments with modeling is proving to be fruitful
in studying carbohydrate structure and conformation.
Speakers at the meeting highlighted the increasing number of
computer programs specially tailored for carbohydrates. "The carbohydrate
chemists are certainly making good use of the increased computer
power and improved software," remarked Alfred
D. French, a carbohydrate chemist at the Department of Agriculture's
Southern Regional Research Center in New Orleans.
Numerous hurdles face the carbohydrate modeler. For example,
the binding sites of proteins and their matching ligands tend
to be seen as locks and keys. Not so with carbohydrates. "Since
they're flopping, the phenomenon of recognition is no longer easy
to think about," said Robert
J. Woods, associate professor of biochemistry and molecular
biology at the University of Georgia, Athens, and organizer of
the ACS symposium.
THAT FLOPPINESS also makes carbohydrates difficult
to crystallize, so their structures are frequently elucidated
with NMR, rather than the X-ray crystallography traditionally
used for proteins. NMR structures represent averages, however,
and it's difficult to say which conformational families are biologically
most important. Models can help narrow down what shapes the carbohydrate
is likely to adopt in a given environment.
The glycosidic oxygens that link together monosaccharides are
the locus of much of carbohydrates' flexibility. These motions
are described by changes in the internal torsion angles (the rotation
angles between the glycosidic oxygen and its two neighbors). The
carbohydrates don't move around freely, of course, but alternate
between several preferred conformations. In fact, in a big carbohydrate,
torsion angles may change while the molecule maintains its overall
shape. "Carbohydrates show a lot of correlated internal motion,"
Woods said.
Carbohydrate motion lends itself particularly to molecular
dynamics simulations--predictions of how the molecule moves over
time. These low-frequency motions can require simulations of 10
to 100 nanoseconds--an expensive eternity in computer simulation
time--compared with protein simulations only a few nanoseconds
long.
Molecular dynamics simulations often involve modeling a molecule
in a "box of water," where it can roll around, buffeted by its
environment. But when a lengthy, floppy carbohydrate tumbles during
the simulation, it's likely to bump into the wall of the box.
To avoid those collisions requires a huge box containing mostly
water, said Woods.
The importance of water in carbohydrate behavior now seems intuitive,
Woods explained, but in the early days, researchers tended not
to include water in their simulations. "It set the field apart
from biomolecular modeling for a while because the simulation methods
weren't developed by theoreticians but by experimentalists who
wanted a simple model for interpreting their data," Woods said.
In biology, however, many carbohydrates are covalently bound
to proteins. And as researchers began to examine this complication,
"it became obvious that they had to include water," Woods said.
Now, a lot of work is devoted to developing ways to treat water
in simulations. For example, is it best to treat the water molecules
explicitly, which costs more in computer time and power, or to
treat water as a continuous background?
Brady's group has pioneered the study of how carbohydrates
interact with water. With their strongly hydrogen-bonding OH groups
and non-hydrogen-bonding aliphatic CH groups, carbohydrates have
a complex relationship with solvents. "In many cases, there's
a direct opportunity for the water to react," Brady said. For
example, water can affect ring flipping in monosaccharides, or
a water molecule bridging parts of the carbohydrate could affect
energetics. Brady is working to bolster his theoretical studies
with neutron diffraction experiments with isotopically labeled
sugars.
A number of speakers at the meeting demonstrated that modeling
is already having a direct impact on the study of biologically
important processes involving carbohydrates.
The bacterium that causes tuberculosis, Mycobacterium tuberculosis,
has one of the more impenetrable coatings known. It resists
the cell-wall-piercing actions of antibiotics and can even survive
inside immune cells. It's known that this impermeability stems
from a close-packed layer of mycolic acids on the outside. These
lipids are attached to the cell membrane through a polysaccharide.
What's unusual is that this polysaccharide is made up of monosaccharides
in the five-membered, or furanose, ring form. This configuration
is higher in energy than the six-membered-ring pyranoses found
in mammalian polysaccharides.
So why did these bacteria evolve to produce polysaccharides
composed of monosaccharides in the least stable ring form? One
hypothesis holds that the bacterium uses furanoses because they're
more flexible than pyranoses, which allows the lipids to form
their tightly packed arrangement. It's analogous to a linker made
of rubber, rather than brick, said Todd
L. Lowary, chemistry professor at the University of Alberta,
Edmonton. But until recently, that hypothesis hadn't been put
to the test, experimentally or computationally.
Now, Lowary--with colleagues Christopher
M. Hadad, chemistry professor at Ohio State University, and
assistant professor Justin B. Houseknecht at Centre College, Danville,
Ky.--is using computational methods to examine the effects of
mycolic acids on the polysaccharide conformation. They selected
a disaccharide fragment and its attached mycolic acids and performed
simulations, randomly generating about 185,000 different conformations.
They then calculated the energies of each conformer, which
gave them insight into what furanose ring conformations were most
likely. The result, they found, is that the lipids do dramatically
influence the carbohydrate's conformation. "They force the sugars
to adopt a conformation they normally wouldn't want to adopt,"
Lowary said.
Though the study is preliminary, he noted, "This idea of a
flexible scaffold now has some support, whereas before it was
just an idea."
Group B Streptococcus is another nasty bug, responsible
in particular for life-threatening illnesses in newborns. Scientists
are working to develop vaccines against group B strep. Such a
vaccine may contain a piece of the bacterium's carbohydrate capsule
that's recognized by the immune system, stimulating the immune
system to form antibodies against the bacteria. A thorough understanding
of group B strep's carbohydrate coating itself is key to understanding
how the antibody interacts with the bacterial surface.
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STACKED An acylated anthocyanin gets its
color stability from the stacked flavylium chromophore (pink
and yellow) and acyl group (blue).
COURTESY OF KAREN WELCH |
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CURRENT THINKING has held that, in general, the
carbohydrate conformation recognized by an antibody--the epitope--can't
contain more than six carbohydrate residues. Anything bigger, and
the antibody can't interact with it.
But in the case of group B strep, the epitope appears to contain
about 20 saccharide residues--which didn't fit in with the reigning
hypothesis. To resolve that discrepancy, it was thought that the
polysaccharide at some critical length changes its conformation,
and that a smaller piece of that new shape is what the antibody
recognizes.
Now, Woods's group has performed dynamics calculations on the
carbohydrate-antibody system that contradict that notion. "We
found that the antibody does indeed interact with a very large
piece [of the carbohydrate]," Woods said. The segment forms a
large corkscrew shape, the inner surface of which is recognized
by the antibody. In fact, the polysaccharide needs to be as large
as it is to form a helix. "So there's no great change in conformation
that occurs," Woods said. "It's unproven experimentally, but strongly
suggested by all simulations."
With knowledge of what's required generally for an antibody
response comes the possibility of thinking about developing vaccines
that protect against more than one strain of the bacteria. "If
you understand the shape dependency, you can see if there's a
common core feature you could exploit," Woods said.
A more biologically benign topic centered around anthocyanins,
carbohydrates that are responsible for much of the red and orange
coloring found in nature--for example, in grapes, strawberries,
and flowers. Scientists are interested in these compounds because
not only do they have antioxidant properties but they could also
serve as natural food colorings, replacing some of the artificial
dyes used currently.
The red color in anthocyanins is due to an aromatic flavylium
cation that exists in an acidic environment. But at higher pH,
the cation is attacked by water, generating a colorless hemiacetal.
That makes anthocyanins useless as food colorings in their current
form.
The color of anthocyanins with an attached aromatic acyl group
tends to be stabler than that of its unacylated counterparts at
higher pH. NMR studies have indicated that this is because the
flavylium chromophore and the acyl group stack with one another,
possibly making water less able to attack the flavylium cation.
AT THE MEETING,
Karen T. Welch, a postdoctoral researcher in the chemistry
department at the University of Tennessee, described her efforts
to determine the characteristic collection of functional groups
that impart color stability--with an eye toward designing useful
food colorings.
Welch simulated the dynamics of a monoacylated anthocyanin
produced by the wild carrot, taking 'snapshots' of different structures.
She calculated the energies of about 1,000 different conformations,
clustered into 18 families, eventually narrowing them down to
two likely candidates, one of which agreed with all the NMR data.
This model, Welch said, could be used to optimize the color stability
of other anthocyanins.
Organic chemistry professor Göran Widmalm of Stockholm
University, in Sweden, and coworkers explored the dynamics of
lacto-N-neotetraose, a carbohydrate that is produced in
humans and also is found in the coat of Neisseria meningitides,
the bacterium responsible for meningitis. Their simulations picked
out two significant conformations for the molecule, which NMR
experiments also identified.
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CROWDED DEFENSE Lipids on the surface of
the tuberculosis-causing bacterium can pack tightly together,
thanks to the flexibility of a carbohydrate scaffold.
COURTESY OF TODD LOWARY |
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Widmalm's group also used the combination of NMR and modeling to
study the binding of a complex oligosaccharide to wheat germ agglutinin
(WGA). Some of the structural variations in the carbohydrate are
characteristics of surfaces of metastatic cancer cells, and so
might be markers of malignancy. WGA's ability to bind with these
carbohydrates is of potential use in developing cancer diagnostics.
The Stockholm researchers used molecular modeling to study
the interaction between WGA and the carbohydrate, which, Widmalm
said, gave them six possible binding modes. They performed NMR
experiments on the bound complex and compared those data with
the model, picking the binding mode that best fit. "The modeling
gave us different alternatives, which we could discard or confirm,
showing the strength of using NMR and simulation together," he
said.
Despite the success of the NMR-modeling combination, there
remains a paucity of experimental data on 3-D carbohydrate structures
in solution, said chemistry professor Anthony
S. Serianni of the University of Notre Dame. His group is
developing NMR methods to establish standard relationships between
experimental data and carbohydrate structure.
In addition to developing NMR experiments, they're using the
computer programs Chymesa and Glyfit, written by Serianni's posdoc
Christophe Thibaudeau. The software helps them decide which conformational
model is consistent with the observed data.
Perhaps the biggest computational effort has been in the development
of force fields for carbohydrates. Force fields are used to calculate
molecular energies and configurations. Chemists can then augment
these force fields to deal with the particular characteristics
of carbohydrates.
"It will be interesting to see how these force fields will
converge in the future to describe the conformation and flexibility
of carbohydrates," Widmalm said. |
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