Chemistry Reference
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Fe 3+ products from the active site toward the nanocage. The interior of the four-helix
bundle is identified as the functional channel based on the observed paramagnetic
effects on residues lining the internal face of the four-helix bundle. The NMR data
provide a basis for the pathway of iron from the ferrous/dioxygen oxidoreductase
site to the central cavity of ferritin [ 164 ]. Such studies open new avenues for the
application of 13 C direct detection experiments to systems with molecular assemblies
larger than 100 kDa.
5 Perspectives
Since the first protein solution structure was determined by high resolution NMR
spectroscopy about 25 years ago [ 166 ], NMR has been established as the only
experimental method that provides both structural and dynamical information at
atomic resolution close to physiologically relevant conditions. Protein structure
determination in living cells has also been achieved recently by in-cell NMR [ 18 ].
However, the limitation of this technique in structural studies lies in low sensitivities
and poor resolution when the size of macromolecules increases. Some proteins, in
particular metalloproteins, might not be stable for a period of time (days or weeks) or
have limited solubility. Moreover, new challenges in life science have also promoted
development of new NMR methods which will improve sensitivities and reduce
acquisition times to fulfil the requirement of characterization of these proteins and
their complexes.
Enormous effort has been made to improve NMR instrumentation in terms
of experimental sensitivity, which results in availability of high-field magnets,
cryogenically cooled probes. In the meantime, tremendous advances in methodo-
logy have contributed to an increased interest in the study of molecular systems of
increasing size and complexity. The introduction of a nonlinear sampling scheme
(instead of conventional uniform sampling) allows a very fast acquisition of multi-
dimensional NMR [ 167 - 169 ]. Many schemes have been developed to reduce the
spectral dimensionality and thus to speed up the experiments, which enables quick
assignment of large proteins [ 170 , 171 ]. The examples include the G-matrix Fourier
transform (“GFT”) NMR approach where sub-spectra from joint sampling of indirect
dimensions are linearly recombined and analyzed [ 172 ]. In the projection reconstruc-
tion (“PR”) method, the corresponding full-dimensional spectrum is reconstructed
[ 171 , 173 , 174 ]. Moreover, various ultrafast NMR techniques including SOFAST/
BEST NMR [ 175 , 176 ] and Hadmard NMR [ 177 ] are also available for studying
biomolecules even in real time. All these new schemes deliver appreciable improve-
ment in the speed of data acquisition and show promise for speeding up multidimen-
sional NMR of normal size proteins [ 170 , 178 , 179 ] and very large proteins [ 180 ,
181 ] as well as sequence assignment for intrinsically unstructured proteins [ 178 ].
13 C NMR spectroscopy is emerging as a powerful tool to complement 1 HNMR
spectroscopy in the investigation of biomolecules, in particular for large molecules
and paramagnetic metalloproteins and also for the study of short-lived molecules
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