Biomedical Engineering Reference
In-Depth Information
Q is a complex function and is provided in Korzhnev et al.
25
When Dv
H
is
zero the imaginary part of l
1
approaches zero and Re(l
1
) reduces to the
Carver-Richards expression for single-quantum dispersion data. The above
relation has an interesting feature not present in the Carver-Richards
formalism, in that it is possible for R
2,MQ
(1/t
cp
) to decrease with increasing
t
cp
in
the
case
of
slow
or
intermediate
exchange processes,
resulting
in
downward curvature in the dispersion plots.
Remarkably, Kay and co-workers have used the
13
C methyl-TROSY
CPMG dispersion experiment to characterise motions in an 82 kDa enzyme
malate synthase G,
58
at temperatures where this enzyme has a rotational
diffusion time, t
c
5 118 ns and in the 300 kDa ClpP protease (t
cp
.400 ns).
61
7.4 Isotopic Labelling Strategies
For large proteins, deuteration of non-solvent exchangeable sites is required,
which increases the sensitivity of the NMR experiment by reducing remote
sources of relaxation. Additionally, in the methyl-TROSY dispersion
experiment, deuteration is essential because external
1
H spin-spin interactions
with the methyl group can give rise to spurious dispersion curves even in the
absence of conformational exchange.
58
For
15
N CPMG or R
1r
dispersion experiments, no other special labelling is
required aside from the normal
15
N incorporation in the growth media.
However, the application of the
13
C CPMG to side-chain methyl groups
requires that those moieties are
13
CH
3
in a molecular background that is
otherwise universally
12
C and
2
H populated. Strategies exist for
13
CH
3
labelling almost all side-chain methyl groups. Perhaps the most widely used is
the so-called ILV labelling strategy, which labels methyl positions of isoleucine
(d1), leucine and valine using metabolic precursors.
62
In the case of leucine and
valine, only one of the equivalent methyl groups is
13
CH
3,
while the other is
12
CD
3
. ILV labelling is achieved by adding a-ketoacid precursors that are
protonated and
13
C-labelled at position 4 to a
2
H,
12
C growth medium prior to
induction. Similar approaches have been developed for specific labellingof
alanine
63
residues. It is also possible to combine these labelling strategies. For
example, ILV and Ala labelling can be combined with minimal spectral
overlap.
64
Both the
15
N and
13
C experiments have been applied to the study of
many large proteins and enzymes. Below we focus on applications in our lab to
the
enzymes
imidazole
glycerol
phosphate
synthase
and
triosephosphate
isomerase.
7.5
Applications
7.5.1 Imidazole Glycerol Phosphate Synthase
Imidazole glycerol phosphate synthase (IGPS) is a 52 kDa heterodimeric
enzyme that exhibits V-type allostery. This enzyme contains two active sites
