DNA-Nanotube-Enabled NMR Structure Determination of Membrane Proteins - DNA Nanotechnology: From Structure to Function - page 327

calculations. Membrane proteins are not generally soluble in aqueous solutions and
need to be solubilized in detergent to satisfy their high hydrophobicity. The resulting
protein-micelle complex can effectively be three to five times larger than the protein
itself and tumbles slowly solution. This leads to rapid transverse relaxation rates,
which broaden the resonance line widths and dramatically complicate the NMR
spectra. However, over the past decade, there has been significant progress in
sample-preparation protocols for membrane proteins; these techniques now enable
studies of much larger structures through the application of transverse-relaxation-
optimized spectroscopy (TROSY) [ 24 ]. The classical paradigm for protein structure
determination by solution-state NMR spectroscopy is to extract and assign a dense
network of 1 H- 1 H NOEs in order to define the three-dimensional fold of a protein.
This approach still presents great challenges for liquid-state NMR-based structural
investigations of membrane proteins, especially on proteins with an -helical

secondary structure, which tend to have a narrower chemical shift dispersion,
large line widths, and poor diversity of the amino acids in transmembrane regions
compared to that of proteins comprised of

'

-sheets, which contributes further to
signal overlap. An alternative to 1 H- 1 H NOEs as a route to high-resolution structural
restraints is found in the controlled reintroduction of anisotropic residual dipolar
couplings RDCs [ 25 - 27 ].

“

16.3 Residual Dipolar-Coupling Restraints

First introduced in 1997 by Tjandra and Bax, the RDC method has become a useful
and almost routine tool for accurate solution-state protein structure determination
[ 26 ]. RDCs constitute an excellent source of structural and dynamic information.
The method depends on the partial orientation of proteins by a liquid crystalline
medium, thus introducing orientation information of magnetic dipole-dipole inter-
action vectors within a common reference frame. The dipolar coupling between two
atoms, j and k, or D jk , is related to the internuclear distance r jk (typically known
in advance (e.g., bond length for covalently linked nuclei)), the angle between the
vector connecting the interacting nuclei and the static magnetic field by the relation
< 3cos 2

, where the brackets indicate time-averaged sampling (Fig. 16.1 b).
These couplings can be a valuable source of angular structural data for NMR
studies of macromolecules because direct information on the orientations of the
corresponding bond vectors relative to the protein's magnetic susceptibility tensor
is provided. However, random molecular tumbling averages these interactions to
zero in conventional isotropic solutions [ 28 ]. It has been shown that RDCs can
be measured by utilizing some type of anisotropic media to allow for partial
alignment of macromolecules and consequently obtain non-vanishing dipole-dipole
interactions [ 26 ]. Such incomplete directional averaging of macromolecules in
liquid crystalline media would allow for the routine measurement of RDCs while
retaining conditions essential for high-resolution solution-state NMR (i.e., rapid
tumbling) (Fig. 16.1 c). For proteins, a highly effective anisotropic media would

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