Chemistry Reference
In-Depth Information
of interactions, so that no signifi cant displacement of the side-chains is required
for sugar binding. Some of these water molecules strategically occupy the posi-
tions that will accommodate the key hydroxyl groups involved in the recognition
(Figure 13.3b) [4]. Similarly, the polar groups of the sugar are expected to be
extensively hydrogen bonded to water molecules. Water molecules directly bonded
to protein and sugar groups show restricted motion compared to those in the bulk
and cannot freely adopt the most favorable orientation for hydrogen bonding to
neighboring water molecules. As a result, water molecules surrounding polar
surfaces are perturbed [7]. On the other hand, organized layers of molecules are
formed over the nonpolar patches of both protein and sugar surfaces. It is clear
that for complex formation water molecules solvating the contact surfaces of
protein and sugar have to be released and returned to the bulk, where they estab-
lish new water-water interactions. This reorganization of the solvent, involving a
large number of molecules, contributes signifi cantly to the binding thermodynam-
ics (see below). The importance of solvent reorganization refl ects the dynamism
of protein-carbohydrate recognition systems, far from the static picture provided
by crystal structures. Another aspect to be considered in this context is the fl exibil-
ity of carbohydrates (please see Chapter 2), which engenders a repertoire of con-
formations in solution. The question arises whether proteins recognize selectively
a distinct conformer, that is, perform conformer selection.
13.3
Selection of Carbohydrate Conformers by Proteins
Nuclear magnetic resonance (NMR) spectroscopy is the key technique for unravel-
ing the three-dimensional structure of biomolecules in solution. Most of the basic
NMR parameters can be employed to monitor the binding of potential ligands to
their putative receptors and, moreover, to provide well- defi ned information on the
mode of binding, including the three-dimensional shape of the bound conformer.
In some favorable cases, by combining state-of-the-art protein labeling techniques
with modern instrumentation, the complete three-dimensional structure of the
protein-sugar complex can also be derived by NMR methods [8].
Information on the bound-state conformation of the carbohydrate ligand can be
obtained by transferred ( TR ) nuclear Overhauser effect ( NOE ) spectroscopy [9] .
The NOE provides information on the distances between different proton pairs of
a molecule. The basis of TR-NMR experiments is that carbohydrate- binding pro-
teins and their small carbohydrate ligands have distinct physical and spectroscopic
properties. In the complex, the bound ligand effectively adapts to physical proper-
ties of the protein and develops strong NOEs. They can be observed when they are
transferred to the sharp resonances of the free ligand (Figure 13.6). Therefore, the
experiment relies on the exchange of all ligand molecules between free and
bound states; consequently, the binding kinetics, particularly the off-rate relative
to the experiment time, are important. In addition, there is an optimal carbohy-
drate ligand to protein ratio for the observation of TR-NOEs, typically using a low
