Biomedical Engineering Reference
In-Depth Information
any case, the relevant functionality of the mutants needs to be confirmed.
Alternatively, lanthanide-binding motifs can be added to the protein, typically
fused to the N or C-terminus or again via a cysteine. In this case, the diamagnetic
reference is provided by the lanthanides La
3+
,Y
3+
,Lu
3+
or Sc
3+
which are
diamagnetic; Gd
3+
on the other hand shows only a PRE effect, useful if PCSs are
not desired.
198
Ma and Opella demonstrate the addition of the calcium-binding
EF-hand motif to the 81-residue membrane-associated, single transmembrane-
helical Vpu protein from the HIV genome; in the absence of Ca
2+
ions, this motif
can bind lanthanides, used in this study to obtain RDC measurements.
199
Zinc-
finger motifs binding cobalt or manganese ions
200
and calmodulin-binding motifs
binding lanthanide ions
201
have also been reported. Greater flexibility to position
the paramagnetic ion is available using thiol-linked tags, similar to the nitroxide
labels used to measure the PRE effect.
202-204
Flexibility of the tag must be
minimised in order to avoid conformational averaging of the magnetic
susceptibility tensor of the metal, which may eliminate the RDC and PCS effects.
Solutions include linking to two attachment sites on the target molecule,
205
or by
using particularly bulky tags.
198,203,206
By constraining the flexibility of the tags,
significantly increased PCS and RDC effects can be observed; for example
Haussinger et al.
206
observe PCS .5 ppm and RDC effects .20 Hz, making such
tags potentially much more useful for large proteins, since the PCS effect is
predicted to be observable at up to 50
˚
. A potential future development is the use
of unnatural amino acids combined with cell-free expression (Section 12.2.5) to
site-specifically incorporate lanthanide labels.
207-209
Applications to membrane proteins have already demonstrated the value of
PRE restraints, for example in determining the structure of the b-barrel,
OmpA. Spin-labels were attached singly at 11 different sites in the periplasmic
turns and b-barrel domain of OmpA, providing 320 PRE restraints.
197
Inclusion of the PRE restraints increased both the accuracy and precision of
the structure, and even on eliminating all the NOE distance restraints and
hydrogen bond restraints, the lowest energy conformers still folded into the
correct b-barrel form.
197
As previously reported, the best correlation between
experimentally derived distances using the PRE restraints and calculated
distances is between 15 and 24
˚
.
197
PREs have also been used in the case of
phospholamban to resolve the degeneracy of structures based on RDCs
210
and
in the structure determination of DAGK
17
and DsbB.
16
Recently a study has
reported a procedure to predict optimally positioned PRE sites in order to
achieve the correct initial fold, using the minimum number of mutations.
211
It
is clear that PRE data is a powerful tool for the structure determination of
membrane proteins, and as PCS tags become more developed it is expected
that they will play an increasingly important role.
12.5.2 Residual Dipolar Couplings
Rapid molecular re-orientation in solution leads to the averaging of dipolar
couplings to zero. However, through the introduction of a finite degree of
