Biomedical Engineering Reference
In-Depth Information
protons
94,95
and a whole suite of solution-like 2D and 3D spectra can be
recorded successfully.
96
An alternative is to use deuteration with 100% back-
exchange of NH groups in combination with fast spinning at 40 kHz in order
reduce
1
H-
1
H dipolar couplings.
97
So far, the studies using
1
H-detection have
centred on small microcrystalline model proteins. Linser et al. show
1
H spectra
of fibril and membrane protein samples, but their sensitivity and resolution is
more limited compared to that of the microcrystalline SH3 domain.
98
Further
optimisation is likely to be required before full resonance assignments and
structures
can
be
obtained
from
proton-detected
spectra
of
membrane
proteins.
13.9 Ultra-Fast Spinning
The use of ultra-fast spinning frequencies of up to 65 kHz has become possible
due to recent hardware developments which have reduced rotor sizes down to
diameters of 1.3-1.6 mm. At these fast-spinning frequencies the
1
H-
1
H dipolar
couplings start to average out which results in significantly narrower
linewidths and means that high-power proton decoupling is no longer
required. Although the small rotor size limits the sample volume to around
1-3 mg of protein, the improved filling factor and narrower linewidths make
up for the reduced amount of sample and good-quality 2D spectra have been
reported for several microcrystalline peptides and proteins.
99-101
The spin dynamics are altered at ultra-fast spinning frequencies comparedto
the more conventional MAS frequencies of 10-20 kHz and low-power
conditions can be used for cross-polarisation and mixing sequences.
101-103
The
use of low-power conditions throughout the NMR experiment, including
proton decoupling during acquisition, means that long inter-scan delays are no
longer required for temperature equilibration of the sample. If the sample
contains or is doped with paramagnetic ions
104
the T
1
relaxation time is
reduced and the inter-scan delay can be shortened even further to values as low
as 200-500 ms
100,101,105
and the sensitivity per unit time is further increased.
Care must, however, be taken with respect to sample temperature and integrity
because the fast spinning itself causes significant sample heating which must be
adequately compensated for with cooling gases.
Ultra-fast spinning experiments promise to be particularly useful for
proteins with limited expression yields, such as many membrane proteins,
since even small sample quantities can give rise to sensitive, high-quality data.
13.10 Dynamic Nuclear Polarisation
Dynamic nuclear polarisation (DNP) involves the transfer of magnetisation
from electrons to protons in order to provide signal enhancement. Although
the DNP effect has been known for many years,
106
it is only over the last 15
years that it has been applied to biomolecules,
107
and in the last few years that
DNP equipment has become commercially available and thus more wide-
