Peptide-Based Drug Design

NMR of Peptides 91
limiting the size of molecules that can be studied. Nevertheless, T2 effects can
be used to detect binding events, since the relaxation properties of ligands are
different from macromolecular targets. Upon binding, ligand resonances are
broadened compared to the free state (41).
15 Nand 13 C labeling of peptides facilitates the study of their molecular
dynamics in solution by measurements of relaxation parameters (42,43).
Heteronuclear relaxation times and heteronuclear NOEs are predominantly
affected by the dipole–dipole interaction of the heteronucleus with the directly
attached proton. Since the internuclear (i.e., chemical bonding) distances are
known from the molecular geometry, correlation times for overall and internal
motions can be determined.
3.5. Signal Intensities
The intensity of a signal in an NMR spectrum is proportional to the number
of nuclei contributing to that signal. Intensity information is thus used implicitly
during the spectral assignment process, but is also valuable when titration experiments
are carried out— i.e., it is useful to determine the mole ratios of species
present when studying protein–ligand interactions. In yet another application the
intensity of signals from amide protons can be used to monitor exchange rates
when the peptide is transferred from H2O toD2Oand thereby deduce information
about internal hydrogen bonding. Attenuation or loss of the amide proton
resonance is an indicator of a solvent accessible amide, while protons that are
not accessible to the deuterated solvent are not exchanged (44–48). Signal intensities
are also employed in the analysis of saturation transfer difference NMR
spectra (Subheading 4.4.2.).
4. NMR Techniques
4.1. Overview
Having introduced the key NMR parameters, we now describe in more
detail how NMR can contribute to the drug-design/-development process.
Figure 1 summarizes the ways in which various types of NMR experiments can
contribute. We have arranged the diagram to reflect three elements: namely the
peptide, the receptor, and their binding interaction.
The simplest experiments are those that focus just on the ligand. These are
typically used to determine solution conformations or 3D structures of ligands.
Homonuclear 1H 1D or 2D NMR experiments are used mainly here. At the other
end of the scale, experiments to study the macromolecular binding partner often
require labeled protein and multidimensional NMR methods, as indicated on the
right-hand side of Fig. 1. Finally, many NMR experiments provide information