Biomedical Engineering Reference
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chain is easily extended by inspecting the nitrogen dimension, which displays
another (v(C a i ); v(C a i )) correlation at v(N i+1 ). At this nitrogen frequency, C a i
is correlated to its successor, C a i+1 . Unlike the 2D NCA experiment, which
exclusively relies on nitrogen chemical shifts for sequential connectivities, 50 the
CANCA experiment can establish sequential assignment via both C a and N
nuclei, eliminating ambiguities in the case of nitrogen chemical shift
degeneracies. Details of the pulse program and results can be found in the
original literature. 51
We also showed that alternate 13 C- 12 C-labelling enables direct correlations
between C a nuclei of neighbouring amino acids via weak long-range 3 J
coupling (y2 Hz). 71 In uniformly 13 C-labelled samples, these weak couplings
are usually masked by the strong 1 J carbon-carbon splittings but are readily
observed in proteins labelled with the alternate
13 C- 12 C-labelling scheme. The
direct sequential connectivities between C a
3 J(C a i -C a i+1 ) coherence
spins via
transfer
can
be
established
by
a
simple
CACA-TOCSY
experiment
[Figure 2.8(A)].
Figure 2.8(B) shows a 2D CACA-TOCSY spectrum recorded on a 5 mM
sample of the uniformly 2 H 15 N and alternately 13 C- 12 C-labelled B1 domain of
protein G (GB1) in D 2 O. The C a - C a i+1 correlations of the protein become
obvious in spectra with mixing times $50 ms [Figure 2.8(C)]. At 132 ms, all
expected inter-residue signals are observed, except for those cross-peaks that
overlap with diagonal peaks and those originating from Leu residues that are
not 13 C a -labelled with the 2- 13 C-labelling scheme. Interestingly, at a mixing
time of 251 ms, 'supra-sequential' C a i -C a i+2 correlations are observed for
y40% of possible residue pairs and even a C a i -C a i+3 correlation was detected.
This represents a unique feature of the CACA-TOCSY spectrum. It is worth
noting that the effective relaxation rate in TOCSY mixing time is the weighted
average of transverse and longitudinal relaxation time (1/T eff 5 W t /T 2 + (1 2
W t )/T 1 ). This might be particularly important for large-molecular-weight
systems in order to take advantage of the long T 1 of deuterated 13 C a . In that
sense, using a spin-lock scheme that has lower W t would generally be favoured.
The build up of long-range C a -C a correlations depends on the 3 J(C a i -C a i+1 )
coupling values as well as the C a relaxation rates. The 3 J(C a i -C a i+1 ) coupling
constants are related to the y i angle, albeit not by a Karplus-type relation. 72
The mean 3 J(C a i -C a i+1 ) coupling constants reported for b-sheet secondary
structure were 1.7 ยก 0.1 Hz, while they were substantially smaller for a-
helices. 72,73 Thus, the CACA-TOCSY experiment provides qualitative
information on the y torsion angle by distinguishing larger 3 J(C a i -C a i+1 )
values for b-sheets from smaller values for helical or loop conformations
[Figure 2.8(D)]. Since the COCO-TOCSY experiment provides information on
the w dihedral angle by a Karplus dependence, 74 the complementary use of
CACA with COCO-TOCSY experiments would be particularly interesting to
obtain structural information from perdeuterated proteins.
On average, 2D CACA-TOCSY was found to be more efficient in
correlating sequential C a -C a correlations than the 2D CA(N)CA experiment,
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