The Use of Residual Dipolar Coupling in Studying Proteins by NMR - NMR of Proteins and Small Biomolecules - page 53

switched [ 28 ]. In this case, it is the electrostatic interaction between alignment
medium and protein that determine the uniqueness of the alignment tensor. Never-
theless, G-tetrad DNA allowed RDC measurements on the fusion peptide of the
influenza viral hemagglutinin solubilized in DPC micelles [ 36 ].

3.3 Collagen Gel

Collagen proteins, abundant in mammals, are made of trimer of polypeptide chains.
Each chain is rich in proline or hydroxyl-proline, glycine, and others, which
together form an extended left-handed polyproline-II structure [ 37 ]. Three copies
of such chains form a right-handed helix, carrying a weak magnetic moment. For
in vitro use the rat tail tendon type I collagen monomers were prepared [ 38 ] and
stored in acidic buffer at 4 C. At pH 6-8 and temperatures over 30 C the collagen
helices will polymerize and cross-link through amino acid side chains, e.g., lysine.
When such reactions occur in the presence of magnetic field, weak alignment can
be achieved [ 29 ]. At a concentration of 13 mg/mL, a sample with collagens
polymerized in the presence of magnetic field yielded deuteron splitting of 20 Hz,
usually sufficient for dipolar coupling [ 29 ].

3.4 Composite Media

As mentioned before, the nature of alignment forces, either steric or electrostatic,
could limit the number of orthogonal alignment tensors we can observe experimen-
tally. However, Ruan and Tolman [ 31 ] showed the interference of the two align-
ment forces could produce one additional orthogonal tensor. They polymerized 5%
(w/v) polyacrylamide gel together with 3-4 mg/mL of bacteriophage Pf 1 in the
presence of the magnetic field. A special tube with a cross section size of 7 5mm

was filled with both media and positioned at a maximal angle of 55 to field B 0
(Fig. 2 ). The gel was dried, later soaked with protein sample, and stretched into
a 5-mm NMR tube. Phages were assumed to be field aligned and trapped at the
direction because their motions were inhibited by polymerized acrylamide. Essen-
tially, the direction of phages formed a tilting angle with the direction of gel stretch.
Proteins were subject to interfered alignment forces, resulting in both strong steric
and electrostatic interactions. Surprisingly, the measured alignment tensors at
different tilting angles are not a simple linear combination of those obtained from
individual alignment using phage or acrylamide gel alone. As reported [ 31 ], at least
three orthogonal tensors were solved for a ubiquitin sample subjected to composite
alignment media. The same group also reported that the unambiguous bond direc-
tion might be determined with only three orthogonal alignment tensors [ 39 ].