Chemistry Reference
In-Depth Information
However, the most important in NMR experiments is that in the exchange
between bound and free states, relaxation properties can be transferred to ligands
bound to proteins Therefore, relaxation properties of small molecules not binding
to proteins differ vastly from those which interact with proteins. While small
molecules are characterized by small transverse relaxation rates, weakly positive
cross-relaxation rates and large diffusion constants, the opposite can be verified
for large molecules. These differences cause line broadening, changes in the
diffusion coefficient and an inversion of the nuclear Overhauser effect's (NOE)
sign.
From these features, ligand-based NMR experiments were developed to verify the
occurrence of intermolecular interactions and identify the region responsible for
this interaction with ligand. In general, three main experimental approaches are
applied. The first one involves magnetization transfer by NOE, which includes the
transferred NOE, NOE pumping and reverse pumping, saturation transfer (STD-
NMR), waterLOGSY, among others [28]. The second and third approaches focus
on changes in relaxation and diffusion behavior of the ligand upon binding to a
protein. In this chapter the most widely used experiments in drug discovery has
been reported. In addition, a detailed description of several methods can be found
in the literature [1, 2, 7, 15, 19, 25-27].
Approaches Involving Magnetization Transfer
Methods using magnetization transfer are the most popular NMR screening
techniques for drug discovery due to several advantages, such as applicability to
large protein complexes, need of low protein concentrations and broad
applicability.
Magnetization transfer can occur between target and ligand spins in transiently
forming complexes through the NOE, which is a manifestation of cross-relaxation
between two nuclear spins close to each other in space. The dependence of NOE
on the spatial relationship of two nuclei makes it an important tool for studying
intermolecular interactions [27]. NOEs connect pairs of hydrogen atoms separated
by less than about 5 Å in amino acid residues that may be far away along the
protein sequence but close together in space [4].
