Biomedical Engineering Reference
In-Depth Information
particularly sensitive to the identity of the following (i + 1) residue, while the
13
C
a
(i - 1) shifts of residues preceding a proline were typically reduced by
more than 2 ppm.
61
In order to avoid steric clashes with proline C
d
and H
d
atoms, the pyrollidine ring skews the distribution of w and y dihedral angles
that can be accessed by the backbone of the preceding residue,
62,63
altering the
population of accessible conformations and thereby changing the chemical
shift. Kjaergaard and colleagues recently pointed out that interactions with
proline are even less favourable if the preceding amino acid possesses a C
b
atom, so using glycine as the reference state for neighbouring residue effects
probably underestimates the correction factors required in most cases.
58
3.5 Detecting Structure and Flexibility
The exquisite conformational dependence of chemical shifts is regularly used to
deduce general properties of protein chains: shifts observed to be close to
random coil values indicate that the protein backbone may be flexible, whereas
significant deviations usually result from the formation of secondary, tertiary
or quaternary structure. The simplest way to highlight these effects is the'Dd
method', where secondary chemical shift values (e.g., for
13
C
a
or
13
C
b
sites) are
plotted against position in the primary sequence [Figure 3.3(a) and (b)].
64
This
logic motivated development of the CamCoils and ncIDP reference states,
which
are
optimised
for
detecting
transiently
populated
structures
in
intrinsically denatured proteins (IDPs) using Dd values.
56,57
Sub-
˚
ngstrom resolution X-ray crystallography reveals rather different
electron density distributions for backbone nuclei in a-helices and b-sheets.
65
As a result, there are clear correlations between secondary shifts and protein
secondary structure, with helical and strand conformations inducing opposite
effects:
13
C
a
shift measurements have higher than average ppm values in a-
helices but smaller values in b-sheets [Figure 3.3(a)], whereas the trend for
13
C
b
shifts is the other way round [Figure 3.3(b)].
66
Calculating the difference
between Dd(
13
C
a
)andDd(
13
C
b
) reinforces this backbone conformation-
dependent effect, while a subsequent three-point smoothing procedure can
minimise additional perturbations due to tertiary structure [Figure 3.3(c)]; the
resulting index, DCAB, is frequently used for preliminary characterisation of
secondary structure in protein NMR studies.
67
Other composite secondary
shift-derived parameters have been proposed, including DCOB
68
and the
'secondary structure propensity' (SSP),
37
but these simple metrics are probably
best deployed as qualitative indicators of secondary structure. Simple methods
have also been developed to highlight characteristic chemical shift signatures
found in b-turns, b-hairpins, edge b-strands and helix capping boxes.
69-72
The chemical shift index (CSI) introduced by Wishart and colleagues
73-75
offers a robust way to mine secondary structure information by jointly
considering
1
H
a
,
13
C9,
13
C
a
,
13
C
b
and
15
N secondary shifts. Threshold values
for different nucleus types are used to attribute one of three states to each
residue. For example: if Dd (
1
H
a
)is,20.1 ppm, the site is assigned to the
