The Use of Residual Dipolar Coupling in Studying Proteins by NMR - NMR of Proteins and Small Biomolecules - page 52

concentrations of alignment media [ 32 ] and application of a new two-stage NMR
tube [ 33 ] have been proposed.

3.1 Charged Polyacrylamide Gel

Mechanically stretched [ 21 ] or compressed [ 22 ] polyacrylamide gel medium is
a promising medium for measuring RDCs on membrane proteins that are
reconstituted in micelles or bicelles. This is due to its inertness and inability to
react with detergents. However, to establish weak alignment that is practical, it is
necessary to polymerize at least 7% (w/v) acrylamide in a sample [ 34 ]. At such
concentration the narrow pore size of the gel matrix limits protein diffusion,
resulting in peak line-width broadening. Meier et al. [ 35 ] initially showed a
sufficient alignment order was achieved by copolymerizing only 2% (w/v) acryl-
amide and acrylic acid, leading to an anionic polymer. Cierpicki and Bushweller
[ 26 ] used acrylamide (

5% w/v) with different charged polymer units to generate
alignment order. In addition to anions, positive charges were introduced by addition
of (3-acrylamidopropyl)-trimethylammonium chloride or N -(2-acryloamidoethyl)-
triethylammonium iodide. With such a charged gel, satisfactory sample stability
and NMR spectra quality were obtained using integral membrane protein OmpA
dissolved in dodecylphosphocholine (DPC) micelles. The RDCs obtained from the
charged gel were directly used for membrane protein structure determination.

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3.2 DNA Based Media

Nucleic acids carry a relatively stronger magnetic susceptibility than proteins and
seldom react with detergent. In the continuous effort to develop detergent compati-
ble alignment media, Douglas et al. [ 27 ] initially exploited DNA nanotubes as
alignment media. Two bundles of six DNA strands in 7,000-base length were linked
through base-pairing sticky ends to form a micron-long DNA filament. The trans-
membrane helices of a T-cell receptor reconstituted in DPC/SDS bicelles were
aligned in a cosolvent of DNA nanotubes at a concentration of 28 mg/mL. The
measured D H-N and D H a -C a were shown to be consistent with the protein structure
determined without RDC.
To ease the DNA nanotube medium preparation and reduce the cost, Lorieau
et al. [ 28 ] used potassium salt of dinucleotide (d(GpG)) that would tetramerize
through guanosine hydrogen bonds at a concentration of 10 mg/mL. The G-tetrad
DNA, similar to bacteriophage Pf 1, is strongly negatively charged. However,
different from phage Pf 1, its liquid crystalline phase is chiral nematic and the
director can run perpendicular to the external field. Analysis of RDCs collected
on a mutant of protein GB3 aligned in G-tetrad DNA showed the obtained align-
ment tensor carried the same directions as those in phage Pf 1, but the sign of D a