Biomedical Engineering Reference
In-Depth Information
that originated in solution NMR are being gradually incorporated into solid-
state MAS NMR approaches. Automated distance restraint assignment
protocols, for example, have been successfully applied to solid-state MAS
NMR data,
67-69
in some cases including additional features, such as the
incorporation of the glycerol labelling scheme.
68
Matrix metalloproteinase-12
(MMP-12) is a Zn
2+
-binding protein whose zinc ion can be substituted by other
metal ions such as paramagnetic Co
2+
. It has been shown that in this case
pseudo-contact shift methods developed for solution NMR can be transferred
to solid-state NMR and used for the structure determination.
70
Similarly, long-
range
distance
restraints
can
be
obtained
from
paramagnetic
relaxation
enhancements (PREs) in spin-labelled proteins.
71
13.6 Dynamics
Solid-state NMR has been used to investigate protein dynamics for many
years, often using isolated isotopic labels as probes.
72,73
More recent studies on
fully labelled protein have shown two distinct approaches. One is to probe
different motional regimes by using different excitation methods.
20
Cross-
polarisation (CP), for instance, acts as a filter for relatively rigid regions of a
protein and only these are visible in CP spectra. J-coupling based excitation,
on the other hand, e.g., using INEPT transfers from
1
Hto
13
Cor
15
N, selects
for highly mobile protein segments, such as long mobile loops or flexible N-or
C-termini. In this way it is possible to record separate spectra, each giving rise
to peaks from separate parts of the protein which are subject to different
classes of motions.
20,21,24,74
In phospholamban, for instance, a distinction
could be drawn between the well-structured lipid-embedded helix (observable
in CP-based spectra) and the flexible exposed N-terminal tail (observablein
INEPT-based spectra).
20
Similar spectra of several rhodopsins have revealed
that some loop regions are highly flexible and visible only in J-based
spectra.
21,24
Line broadening, either through mosaic spread
75
or though spatial
and temporal averaging around defined axes for helical peptides in
membranes, has been simulated to demonstrate distinct spectral features.
76
Such manifestations in heteronuclear correlation (HETCOR) or polarization
inversion spin exchange at the magic angle (PISEMA) spectra, may be induced
by hydration, temperature or simply poor sample alignment, but can be
included in data analysis to give structural and dynamic information.
75-77
An alternative, more quantitative, approach has been to measure
anisotropic interactions and relaxation rates across many sites so as to derive
order parameters.
13
C-
1
Hand
15
N-
1
H dipolar couplings or
13
C and
15
N CSAs
are modified by internal motions so comparison between the measured and
calculated values provides an estimate of internal dynamics. In addition,
longitudinal
15
N relaxation rates can be measured as convenient probes for
internal motions because of the absence of molecular tumbling in the solid
state. Early studies using small numbers of labelled sites
78,79
have been
expanded to the study of uniformly [
13
C,
15
N]-labelled full proteins.
10,80-84
