Peptide-Based Drug Design

100 Westermann and Craik
times and can change from positive, to zero, to negative values depending on
the size of the peptide being studied. The correlation time at which a molecule
displays negative NOEs depends on the temperature and the field strength of the
spectrometer used. As a guideline, peptides around 1 kDa display only weak or
no NOEs on a 500 MHz spectrometer, and ROESY experiments (60) may be
necessary for peptides of this size. Notwithstanding this complication, NOEs are
extremely useful.
4.4.1. Transferred NOE
In this method the different correlation times and NOE build-up rates of free
and bound states of a ligand are used to detect NOEs on the free ligand that
reflect its bound conformation. This class of experiment usually is conducted by
performing transient NOE experiments by means of 2D NOESY spectra. The
basis of the technique is that small molecules show positive NOEs while macromolecules
show negative NOEs and that NOEs build up/decay quickly in macromolecules
but slowly in small molecules. Upon binding of a small molecule to
a macromolecule, the motional characteristics of the small molecule change and
it shows negative NOEs. After dissociation from the complex, information on
the negative NOE is transferred back to the free ligand, which is usually in large
excess and readily detectable (12).
Besides intramolecular transferred NOEs, intermolecular transferred NOEs
are of interest when studying molecular interactions (61,62). In this case NOEs
build up between protons of the ligand during its residence time in the binding
pocket. This gives information on the orientation and conformation of the ligand
in the binding site. The transferred NOE approach for structural characterisation
of the bound conformation of a ligand is schematically illustrated in Fig. 8
Fig. 8. Schematic representation of the trNOE effect. On the left the conformation of
the unbound ligand does not allow the buildup of a NOE because the two protons are too
far away. In the middle representation the bound conformation orients the two protons at
a distance that allows the buildup of a NOE. After dissociation of the complex the information
of spatial proximity of the two protons in the bound conformation is transferred
back into the solution conformation (right), where the NOE still is observable.