Chemistry Reference
In-Depth Information
to structure determination. In addition, some smaller membrane proteins are
comprised of structural elements that only weakly interact with each other, if at
all, and would therefore not be expected to give rise to long-range NOEs. Regard-
less of whether the absence of assignable long-range NOEs is due to structural or
spectroscopic causes, in both cases distance restraints are accessible using
approaches that do not require assignment of side chain chemical shifts or long-
range NOEs. This approach has been used to determine structures for a significant
number of membrane proteins (Table
2
, Method VII), with residual dipolar
couplings (RDCs) and paramagnetic relaxation enhancement (PRE) data being
the primary sources of long-range distant restraints.
5.3.1 Measurement of RDCs
Although rapid molecular reorientation rates in solution are responsible for the high
resolution of liquid-state NMR spectra, this also gives rise to isotropic averaging that
prevents the observation of orientation-dependent phenomena. However, it is possible
to detect these orientation-dependent effects while retaining the advantages of solution
phase spectroscopy by subjecting the protein of interest to a weakly aligning environ-
ment [
311
]. The resulting non-zero averaging allows data such as the RDC and
residual chemical shift anisotropy (RCSA) to be acquired and used for structure
determination.
From a practical viewpoint, the generation of weakly aligned membrane protein
samples poses additional challenges relative to the case for water-soluble proteins.
While the first RDC measurements on water-soluble proteins used liquid-crystalline
phases of bicelles to induce weak alignment [
312
], the extension of this measure-
ment to membrane protein samples awaited the development of detergent-resistant
alignment media. The first, and one of the most widely used, type of detergent-
compatible systems is the aqueous phase of mechanically strained polyacrylamide
gels [
313
]. Asymmetry in gel pores can be straightforwardly created with the
assistance of a commercially available gel-stretching apparatus [
314
], or simply
by applying axial compression to gels with diameters smaller than that of the NMR
tube interior [
313
,
315
]. A specialized type of NMR tube has also been designed
that makes it possible to measure both isotropic and aligned states in the same gel
sample, a particularly important advance for accurate measurement of small
RCSAs [
316
].
Variations in the degree of alignment can be obtained by using different gel
densities, aspect ratios, and gel cross-linking ratios [
313
,
314
]. However, at the gel
concentrations required to maintain mechanical stability of the gel (
4% w/v),
diffusion of large protein-detergent complexes into the pores can be hindered,
reducing the amount of protein that can be introduced into this medium [
68
,
247
].
Consequently, the first RDC measurements on membrane proteins were done with
smaller protein-detergent complexes using higher gel concentrations [
137
,
160
,
314
,
317
,
318
]. More recently it was demonstrated that the ~100-kDa DAGK-DPC
complex could be introduced into a 4% hydrated gel by allowing ~2 days for the
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