Biomedical Engineering Reference
In-Depth Information
Table 1.1
Time requirements for obtaining methyl-group resonance assign-
ments.
Sample information
NMR time
a
Protein
kDa
Oligomer
Number
Preparation time
Ref.
b
60
Proteasome
670
28 (a
7
b
7
b
7
a
7
)
Not stated
26 days
4
b
6 weeks
c
45
ATCase
300
12 (r
6
c
6
)
29 days
64
b
2 weeks
d
27
TET2
468
12
3 days
a
Total reported NMR acquisition time required for assignment of the full-size complex (including
time required for assignment of subunits). For TET2 a single 2D (
1
H,
13
C) HMQC spectrum (max
1 h) was required per mutant. This value does not include time taken to analyse and interpret the
data.
b
Only includes samples with different isotope-labelling schemes (samples of additional
mutants of proteasome not included) or, in the case of TET2, the 64-member library of single point
mutants.
c
This time included optimisation of sample expression and purification as well as the
production of samples with different isotope labelling patterns.
d
Time calculated based on parallel
expression and purification using pre-packed Capto-Q plates (GE Heathcare), without specialised
automated equipment (Crublet et al., in preparation). In initial implementation reported by Amero
et al. (ref. 27), purification of the 64 mutants was performed manually and the total preparation
time was 1 month. Value does not include the time required to produce the library of mutant
vectors, which can be performed using an automated molecular biology platform or purchased
directly.
1.4 High Molecular Weight Protein Applications of
Methyl-Specific Isotope-Labelling
With tractable spectra and accompanying resonance assignments it is possible
to use NMR spectroscopy to address interesting biological questions. When
dealing with proteins in excess of 100 kDa it is not feasible to solve de novo
protein structures, however NMR spectroscopy of methyl groups can be used
to probe local structure, to monitor function (in real-time) and characterise
molecular dynamics on multiple timescales. Longer range structural informa-
tion can also be obtained using residual dipolar couplings or paramagnetic
relaxation enhancement measurements. A non-exhaustive selection of exam-
ples is provide in Table 1.2 to give the reader a taste of the kind of systems that
can be studied and the experiments that can be performed on high molecular
methyl-labelled perdeuterated proteins.
1.5 Conclusions and Future Directions
NMR spectroscopy is often considered a small-molecule technique, ideal for
characterising proteins smaller than 20 kDa. However, as we have detailed
here, this perception is no longer valid. It is now increasingly feasible to apply
solution NMR techniques to protein systems as large as 1 MDa. One of the
main reasons for this raising of the upper size limit has been the development
of protocols and molecules that allow residue-type-specific protonationof
