Biomedical Engineering Reference
In-Depth Information
structure determination today. Selective labelling, e.g., methyl groups (ILV),
15
in an otherwise deuterated protein, together with advanced structure
calculation techniques allow extension of NOE-based structure determination
to about 50 kDa molecular weight.
10,55,128
4.3.2 Residual Dipolar Coupling
Dipolar couplings between two nuclei depend on their distance, and the angle
between the inter-nuclear vector and the magnetic field.
56,57
In isotropically
tumbling molecules the dipolar couplings average out, but in weakly aligning
media a residual dipolar coupling can be measured for nuclei that are close in
space, i.e., bonded like N-H, or within a single peptide plane, like C
a
-H. The
most popular pair is N-H, but N-C, C
a
-H
a
, C-C
a
,C
a
-H or methyl C-H are
also frequently measured.
56
Importantly, combinations like N-H, C-C
a
, N-C
allow the peptide plane to be restrained for each residue.
58,59
By separating out the rotational tumbling motion of the molecule and
assuming that internal motions are restricted to isotropic fluctuations around
an equilibrium orientation one obtains structural restraints of the inter-nuclear
vector within an alignment frame.
57
Given a specific alignment tensor the inter-
nuclear vectors that are consistent with a measured residual dipolar coupling
describe an ellipse on the surface of the unit sphere. This ambiguity can be
reduced by using more than a single alignment, which yields additional ellipses
restraining the inter-nuclear vector to the intersection point(s).
59
4.3.2.1 Advantages
RDCs yield orientational restraints with respect to a common molecular
frame, and thus provide long-range information. This renders RDC data
powerful for docking complexes or determining domain orientations.
60,61
The
interpretation of RDC data is unambiguous in terms of assignments, which is
in sharp contrast to many NOEs which are ambiguous and can only be
resolved during the course of iterative refinement (see above). Thus, RDCs are
popular for cross-validation of NOE-based structures.
Measurement of residual dipolar couplings is more amiable to high
molecular weight molecules than NOESY experiments. RDCs do not require
contacts to restrain tertiary structure which renders them useful for extended
molecules, such as DNA, RNA, membrane proteins.
62
Structural data can be
obtained using backbone atoms only, whose chemical shift assignment using
triple resonance techniques is robust and routine even for high molecular
weight proteins.
12,63,64
In fact, starting from X-ray structures of sub-
components, the domain orientations in the 128 kDa Enzyme I dimer and
its 146 kDa complex with HPr have been solved by a combination of RDC and
solution scattering (SAS) data.
12,64
