Biomedical Engineering Reference
In-Depth Information
correlation spectra recorded of different mutants. The NMR experiment used
is therefore sufficiently simple and sensitive to allow assignments to be made
directly from the full-size protein complex. As an example, 15 out of 17
(
1
H,
13
C) resonance frequencies of methionine e-methyl groups of poliovirus
RNA-dependent RNA polymerase were assigned by site-by-site mutation
strategy.
47
The protein was labelled with [e-
13
CH
3
]methionine and conse-
quently sites with methionine-to-isoleucine mutations lacked an observable
correlation. A similar approach was used to obtain isoleucine d
1
-methyl group
assignments in the protease, Clp2 (ref. 48). A combinatorial approach using
single- and triple-site isoleucine-to-leucine mutations has also been used to
assign [d
1
-
13
CH
3
]isoleucine resonances of the RNA-directed RNA polymerase
of cystovirus phi6 (ref. 49). Mutagenesis has also be used to verify assignments
made using 'divide-and-conquer' style approaches. For example, during the
assignment of the 20S proteasome, significant chemical shift differences
between monomeric and oligomeric samples were observed which prevented
resonance assignments being unambiguously transferred between oligomeric
species.
45
Under these circumstances, assignments were made or confirmed
using site-directed mutagenesis.
The assignment-by-mutagenesis strategy has recently been applied to a
468 kDa supramolecular protein oligomer in a highly systematic and
streamlined way.
27
Using only 2D (
1
H,
13
C) correlation spectra recorded of
wild-type and mutant proteins it was possible to perform complete
resonance assignment of the 34 isoleucine-d
1
and 30 alanine-b methyl
groups of a homododecameric archeal protein, TET2 (ref. 27; Figure 1.1). A
protocol was reported which involved automated mutagenesis robots and
medium throughput, semi-automated protein expression, methyl group
labelling, and purification. Expression culture volumes were minimised to
reduce preparation costs and to simplify the handling of multiple samples in
parallel.
Conceptually, assignment-by-mutagenesis is straightforward. In practice,
however, resonance overlap, missing peaks from the WT spectrum and off-
target changes to mutant spectra can complicate analysis.
47-49
Amero and
colleagues noted that analysing the full library of single-site mutations greatly
simplified the procedure of resonance assignment by allowing tentative
assignments to be cross-validated multiple times with unambiguous ones.
27
1.3.3 Time Frames for Resonance Assignment of Methyl Groups
in Very Large Proteins
Despite some dramatic examples, methyl group resonance assignment in
supra-molecular systems remains time consuming. Table 1.1 provides the
reader with an idea of the lower-end estimates of the timescales required for
methyl
group
assignment
in
large
proteins
using
values
reported
in
the
respective publications.
