Biomedical Engineering Reference
In-Depth Information
natural protein fibres including cocoon and spider silks have been studiedat
lower resolution.
43
13.3 Assignment Strategies
While early assignment strategies were based on 2D spectra,
44
the inclusion of
3D spectra is now widespread. 3D NCACX, NCOCX and CANCO or
CAN(CO)CX spectra have been used frequently in order to obtain sequential
assignments using a backbone-walk approach in which side-chain resonances
are obtained simultaneously.
45,46
More recently, Schuetz et al. have proposed
the use of NCACB, N(CO)CACB and CAN(CO)CA spectra for backbone
walks.
47
An advantage of this strategy is that sequential links are established
using the C
b
rather than the C9 chemical shift, thus making use of the greater
chemical shift dispersion of the C
b
chemical shift. Furthermore, the transfer
types used in the pulse sequences have been selected for optimal efficiencyon
uniformly
labelled
protein.
Side-chain
correlations
are
obtained
from
N(CA)CBCX and CCC spectra.
For large proteins or those with limited sensitivity, the labelling strategy can
assist with amino-acid type identification and sequential linking of residues
48,49
or can provide convenient entry points into the assignment.
50
13.4 Labelling Strategies
Although uniform [
13
C,
15
N]-labelling forms the basis of most solid-state NMR
studies, the use of alternative labelling schemes is becoming increasingly
common. Hong and Jakes
51
and Castellani et al.
1
introduced the [1,3-
13
C]-
glycerol and [2-
13
C]-glycerol labelling schemes
52
to solid-state NMR in order
to obtain narrower linewidths, decrease overlap and increase the number of
long-range distance correlations observed. Such extensive labelling schemes
have since been used both to obtain distance restraints
1,53
and to facilitate
assignment.
48,49
Spectra of large membrane proteins are often characterised by intractable
degrees of overlap for aromatic or hydrophobic residue types which dominate
the transmembrane region. A successful approach for such systems has been
the use of 'reverse labelling'
54
to suppress the labelling of e.g., Phe, Tyr, Val or
Leu residues in an otherwise U-[
13
C,
15
N]-labelled protein.
21,23,24
'Forward
labelling' using uniformly or
2,3
-labelled amino acids has also proved useful but
is a more expensive option.
29,55
Unless cell-free labelling
56
or solid-phase
peptide synthesis is used these amino-acid-specific labelling schemes are
unfortunately constrained by the catabolism and metabolism of E. coli which
may cause a degree of scrambling of the isotopic labels.
Labelling strategies are also important for the detection of intermolecular
peaks. Dilution of uniformly labelled protein with around 70-80% unlabelled
protein
1
has the drawback that signal intensities are significantly reduced and
that the identification of contacts depends on the disappearance of cross-peaks
