Biomedical Engineering Reference
In-Depth Information
13
C detection has proved particularly popular for paramagnetic pro-
teins,
254,255
and has also been demonstrated for the backbone assignment of
a 37 kDa homotrimer without the need for deuteration
256
and for in-cell
NMR.
257
These are all systems which suffer from very fast spin relaxation.
Similar strategies could benefit the study of large membrane protein systems in
particular when solubilised in large membrane mimetics.
12.6.2 Alternative Sampling
NMR studies of membrane proteins are constrained by low-sensitivity,
crowded spectra with broad lines and short sample lifetimes. When using
conventionally sampled spectra that rely on Fourier transformation, it can be
difficult to record experiments with sufficient sensitivity and resolution during
the relatively short amount of time over which the membrane protein sample is
stable. Various experimental schemes have been suggested that, benefitting
from short selective proton T1 values, aim at faster signal-averaging through
rapid pulse repetitions, so called SOFAST type experiments.
258-261
Alternatively, it is also possible to reduce experiment times through the
reduction of the number of data points recorded in the indirect dimensions.
Typically this is achieved by under sampling using a sampling schedule thatis
exponentially weighted, whereby most data points in the indirect dimensions
are recorded at short time points to maximise sensitivity, and then a few points
are recorded at long time points in order to improve resolution and avoid
truncation artefacts.
262-264
Non-uniform sampling precludes use of the discrete
Fourier transform and consequently, a number of methods have been
proposed to reconstruct spectra, including maximum-entropy (ME) recon-
struction,
262-264
multi-dimensional decomposition
265,266
and, more recently,
compressed sensing.
267,268
As a result of reducing the number of data points
sampled, the time saving may be used either to shorten overall experiment
times, particularly useful for unstable proteins, or to increase the number of
scans recorded at short evolution periods, significantly increasing sensitivity
which can be of particular benefit for large membrane proteins. These methods
have been used to study a number of membrane proteins; to date ME
reconstruction has been applied to 3D backbone assignment of pSRII
19,269
whilst multi-dimensional decomposition (MDD) was used in the study of
VDAC-1 to record 4D NOESY experiments.
13
ME has also been used for
other large proteins including EntF.
178
Compressed sensing has been
demonstrated to have some benefits over ME reconstruction, allowing further
under sampling of HN(CO)CA and HNCA experiments recorded on pSRII,
267
and is also predicted to enable under sampling of 3D NOESY spectra which
currently require extremely long acquisition times.
267,268
Such methods have
the significant advantage that they require no new hardware and can be easily
implemented and applied to existing pulse sequences.
