Chemistry Reference
In-Depth Information
Approaches involving Relaxation Time
In NMR spectroscopy the term relaxation is used to describe the process which
restores equilibrium magnetization and random phase [26]. There are two distinct
relaxation mechanisms,
i.e
. transverse (T2) and longitudinal (T1) relaxation with
the respective relaxation rates
R
2 and
R
1. Longitudinal relaxation (T1) is non-
selective and can be very similar for small and large molecules whereas transverse
relaxation (T2) is quite different [39]. Small ligands tumble rapidly in solution
and have small transverse relaxation rates. Large molecules tumble slowly and
relax fast. Hence a ligand, bound to a protein, relaxes faster than the unbound
ligand [4].
NMR relaxation can be associated with fluctuating electrical fields, originated
from the overall tumbling and internal motions of the molecule. The transverse
relaxation rate of a nucleus in a molecule is, to a first approximation, proportional
to its tumbling correlation time (c). Since large molecules tumble slowly in
solution, the c of their rotational motion is relatively long, causing large
relaxation rates
R
2 and therefore large line widths of NMR signals. Compounds
interacting with large molecular weight receptor show broadened lines and
increased
R
2 values. Thus, in order to detect binders, compounds line-shapes can
be compared in the presence and absence of the receptor, since binding-induced
R2 enhancements may be visible by simple line-broadening of proton resonance
lines upon addition of the receptor [4, 25, 39].
Another experimental method involving T2 relies on the separate acquisition of
spectra from the mixture of compounds in the presence and absence of the protein.
Subsequent subtraction leads to spectra containing contribution only from binding
ligands [3]. The potential of these methods for NMR screening has been described
by Fesik and coworkers. They identified a ligand for FKBP [40] with an affinity
of 200 µM from a mixture containing other eight compounds known as FKBP
non-binder [41]. On the other hand, T2-filter can be applied to selectively remove
signals of the bound ligands to proteins in a mixture of compounds [3, 39]. The
increase in relaxation rate can be enhanced tenfold or more by employing spin
label coupled to the protein, allowing a reduction in protein concentration [4, 42].
