Protein Structure Determination by Solid-State NMR - NMR of Proteins and Small Biomolecules - page 197

that trigger the conformational changes are involved in activation and formation
of the G protein binding site. In the last few years, the Smith group has focused
on SSNMR studies of structural changes on the extracellular side of the recep-
tor caused by retinal isomerization. By combined SSNMR chemical shift
measurements and 2D-dipolar assisted rotational resonance (DARR) [ 154 ]NMR
measurements with selective labeling schemes and mutagenesis, they have
published several papers addressing the functions of the displacement of EL2 on
rhodopsin activation. Figure 8 shows the two-dimensional 13 C DARR NMR spectra
of retinal-EL2 interactions - close contact between the retinal 13 C14 and 13 C15
carbons and 13 C b -Ser186 (Fig. 8a), between the retinal 13 C12 and 13 C20 carbons
and 13 C1-Cys187 (Fig. 8b), and between the retinal 13 C12 and 13 C20 carbons and
13 C

-Gly188 in rhodopsin (Fig. 8c). But the contacts between the retinal 13 C9 and
13 C12 carbons and U- 13 C 6 -Ile189 in rhodopsin or Meta II were not observable
(Fig. 8d) [ 18 ]. All the results indicate that the formation of Meta II is accompanied
by the displacement of EL2 away from the retinal binding site and that there is a
rearrangement in the hydrogen-bonding networks connecting EL2 with the extra-
cellular ends of transmembrane helices H4, H5, and H6. This displacement is
coupled to the rotation of TM5 and breaking of the ionic lock connecting TM3
and TM6 [ 18 ]. These comprehensive results may lead to further investigation of the
molecular mechanism of the cavity formation between H3, H5, and H6 for
G protein binding [ 155 ].

a

Fig. 8 Two-dimensional 13 C DARR NMR spectra of retinal-EL2 interactions. Rows from the
two-dimensional 13 C DARR NMR spectra of rhodopsin ( black ) and Meta II ( red ) are shown. The
rhodopsin crystal structure ( gray ) with the Meta II model ( orange ) obtained from molecular
dynamic simulations are shown in the middle of the figure. Adapted from [ 18 ] with permission
from Nature Publishing Group