NMR as a Unique Tool in Assessment and Complex Determination of Weak Protein–Protein Interactions - NMR of Proteins and Small Biomolecules - page 40

unlabeled target. This experiment can detect very weak NOEs, possibly up to 7 ˚

in

distance, due to the deuteration effect.

1.3 Residual Dipolar Couplings

Although NMR-based applications on dipolar coupling have been mainly associated
with solid-state NMR or NMR of oriented samples, they have been recently applied
to solution NMR for studying macromolecular structure and function in an aqueous
solution [ 20 , 21 ]. The dipolar coupling describes a through-space interaction that
arises between any two magnetically active nuclei. It depends upon the distance
between two atoms, which is constant for the nuclei connected by covalent bonds
such as 1 H- 15 Nor 1 H- 13 C, and the orientation of the connecting vector with respect
to the external magnetic field. In solution, dipolar interactions are averaged because
of fast isotropic tumbling. However, if the macromolecules experience obstacles in
some directions due to partial alignment with orienting media, for example, bacterio-
phage, bicelles or polyacrylamide gels, the dipolar couplings are not completely
canceled out and whatever is left is designated as residual dipolar couplings
(RDC). By measuring RDC the orientation of the molecular alignment tensor could
be defined, providing the information about mutual orientation between the domains
within single macromolecule or between binary units of the complex. Thus the quest
began to find robust ways to orient the media weakly without significant increase
in viscosity of the system or generation nucleation points for aggregation. The general
idea is based upon a fact that certain media, possessing sufficiently large magnetic
susceptibility anisotropy, can be aligned spontaneously by high magnetic field. In
earlier 1990s, bicelles, disk-shaped particles made from the lipid/detergent mixtures,
were introduced [ 22 ] for this purpose, followed by rod-shaped viruses [ 23 ], mechani-
cally orientable systems [ 24 , 25 ], and G-tetrad DNA [ 26 ]. As compared to the more
conventional NOEs approach, RDC carry complementary information: while NOEs
provide only local distance restraints, RDC contain long range orientational informa-
tion (e.g., see review by [ 27 ]), thus delivering powerful long-range geometric
constraints for proper subunits orientation during the structure determination of the
complex. In the case of wPPIs that may undergo fast exchange between the bound
and free forms of the binding partners, measured RDC will represent the population
weighted average values of those in bound and free form. Theoretically, knowing the
molar ratio of bound and free forms (from K d and molar concentrations), and after
measuring RDC in the free form, one can calculate RDC in bound form [ 27 ]. From
the RDC of weakly bound subunits, their alignment tensors can be calculated and
matched for defining the structure of a weak complex. One example using this
strategy to determine the structure of weak complex is -methyl mannose bound to

mannose-binding protein with a K d ~1mM[ 28 ]. In practice, however, it is not
always straightforward for small ligands to determine the accurate fraction of bound
form and, thus, full saturation could be beneficial to utilize the RDCs of the fully
bound form.

a