Chemistry Reference
In-Depth Information
for the small peptide bound to its target protein [
16
]. In a nutshell, if the affinity of
the interaction in question is low due to the fast dissociation rate, cross relaxation
between protons of the peptide in the bound state, which is governed by the large
correlation time of the complex, is transferred to the free state through chemical
exchange. This phenomenon is manifested by the appearance of additional peaks
at the original (corresponding to free state) frequencies in the NOESY spectrum of
the peptide when it is mixed with a small molar portion of the large, typically over
50 kDa in molecular weight, target protein. Protein-peptide ratios for trNOESY
may vary from 1:10 to 1:200 with mixing times ranging from 50 to 500 ms. Both
parameters need to be optimized for each particular case. Substantially increased
number of NOEs should be observed for the peptide in the presence of its target
protein comparatively to the peptide free form. The method requires no isotope-
labeling and is suitable for examination of protein-ligand interactions over a wide
range of
K
d
s (micromolar-millimolar) [
7
]. The method can be applied to study
initial lead compounds weakly bound to the target protein, which allows the
structure determination of the bound compounds for further optimization leading
to high affinity binding.
Half-Filtered NOESY (Intermolecular NOEs)
Half-filtered NOESY approach was developed to detect inter-molecular contacts
in the form of NOEs only between protons pairs in which one of the protons is
attached to
15
Nor
13
C nuclei while the other is attached to magnetically inactive
14
Nor
12
C nuclei. Thus it requires the preparation of
15
N-
13
C-labeled protein
mixed with its unlabeled binding partner, and/or vice versa. Two types of half-
filtered NOESY experiment are commonly used [
17
]: (1) three dimensional
13
C-separated-
15
N,
13
C-filtered NOESY, which detects NOEs between protons
attached to
13
C atoms of the doubly labeled protein and those attached to
12
C and
14
N on the unlabeled protein, and (2) three-dimensional
15
N-separated-
15
N,
13
C-
filtered NOESY, which detects NOEs between
15
N-attached and
12
C-,
14
N-attached
protons. However, there are pulse sequences available with smart combinations of
both, when separation in
13
C and
15
N dimensions can be achieved simultaneously,
significantly reducing the experimental time. The sensitivity of this experiment
crucially depends on the lifetime of a protein complex. For weak PPIs characterized
by high k
off
the actual portion of the complex within the sample might not be high
enough for observing the intermolecular NOEs. However, in favorable cases, when
the concentrations of both binding partners are high [
18
], the complexed state could
be detectible with the help of high-sensitivity NMR instruments, such as those
equipped with cryo-probes. Another relatively more sensitive experiment is the
15
N-edited NOESY on a
15
N/100% deuterated protein bound to the target, which
will detect the NOEs between the amide proton of the
15
N-labeled protein and any
nearby protons of the target [
19
]. This experiment can be complementary to those in
(1) and (2) but provides a very unambiguous assignment of the amide protons,
which are usually well resolved in the HSQC spectrum, to the protons of the
