Chemistry Reference
In-Depth Information
influenza B proton channel [
326
] and the
a
IIb
b
3 TM helix heterodimer in isotropic
bicelles [
184
].
With the exception of the strained polyacrylamide gel, many of the detergent-
resistant media described possess significant charge that can give rise to unfavor-
able interactions with the alignment media, potentially broadening spectra beyond
detection [
327
]. An alternative that avoids this problem exploits the significant
anisotropy of the magnetic susceptibility of lanthanide ions such as Yb
3+
and Dy
3+
.
This leads to weak magnetic field-induced alignment that allows RDCs of
lanthanide-bound species to be measured [
328
]. For proteins that do not have a
high-affinity metal-binding site, it is possible to introduce a metal chelator via
covalent modification of a unique cysteine residue [
327
,
329
,
330
] or by engineer-
ing a metal-binding EF-hand as was done for the HIV Vpu protein [
331
] and a
modified form of OmpA [
332
]. However, to maximize the information content from
these experiments, high affinity tags with a single susceptibility tensor that is
minimally affected by dynamics should be used. For this purpose, sulfhydryl-
reactive EDTA based analogs have been designed that, unlike the non-stereospe-
cifically metal-binding precursor, create a single stereoisomer of the metal-bound
complex [
333
].
5.3.2 Applications of RDCs in Structure Determination
The magnitude of the RDC depends on the degree of alignment induced in the
sample, the inter-nuclear distance separating the two atoms involved, the orientation
of the internuclear bond vector with respect to the external magnetic field, and the
local dynamics of the atoms involved [
311
]. When measured for a set of covalently
linked backbone atoms with similar dynamic properties, RDCs can be used to
determine the relative orientation of the corresponding peptide planes. A large
number of NMR experiments are now available to measure these types of RDCs
(reviewed in [
334
]) and inclusion of at least one type of RDC is often performed in
standard protein structure determinations (reviewed in [
335
,
336
]), including those
for membrane proteins [
39
,
152
,
162
,
166
,
326
,
337
,
338
]. However, RDCs alone are
not enough to determine a unique protein fold when starting from an extended
structure, since there is 16-fold degeneracy in the number of peptide plane
orientations consistent with the measured couplings [
339
]. Consequently, RDCs
cannot distinguish between structures of different quality at early points in a structure
calculation when differences between calculated and actual structures are large (i.e.,
rmsd
10
˚
)[
340
,
341
]. RDCs are instead used to refine NOE-based structures via
a scalable energy function that gradually increases the magnitude of the RDC energy
penalty as the NOEs drive the fold closer to its final state [
342
]. This allows the
structure to be oriented into its alignment frame before the couplings refine the
structure, an approach that can lead to an increase in the quality and precision of an
NOE-based structure [
342
,
343
]. However, these benefits are usually only realized
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