Biology Reference
In-Depth Information
crystallography at 3.8
˚
(
Lodowski et al., 2007; Salom, Le Trong, et al., 2006
). Then,
a parallel dimer of
2-AR)/T4L was obtained in which the
interactions between the two monomers were mainly mediated by lipids (
Cherezov
et al., 2007
). More recently, crystals of chemokine CXCR4 receptor (
Wu et al.,
2010
),
b
2-adrenergic receptor (
b
k
-opioid receptor (
Wu et al., 2012
),
m
-opioid receptor (
Manglik et al.,
2012
), and
1-AR (
Huang, Chen, Zhang, & Huang, 2013
) revealed parallel homo-
dimer arrangements with substantial protein-protein interfaces probably reflecting
functionally relevant interactions.
b
24.1
CRYSTALLIZATION OF BOVINE RHODOPSIN
The first crystal structure of ground-state rhodopsin, solved in our laboratory
(
Palczewski et al., 2000
), contained antiparallel dimer interactions and the crystals
were disrupted when illuminated. Next, Schertler's laboratory obtained hexagonal
rhodopsin crystals (
Suda, Filipek, Palczewski, Engel, & Fotiadis, 2004
),
(
Stenkamp, 2008
) also with an antiparallel monomer-monomer arrangement. Then,
through modifications of rhodopsin's purification protocol, our laboratory obtained
two new crystal forms (trigonal and rhombohedral) able to withstand photoactiva-
tion. This permitted the structure of an activated GPCR to be solved for the first
time (
Salom, Le Trong, et al., 2006; Salom, Lodowski, et al., 2006
). Interestingly,
one of the rhodopsin-rhodopsin interfaces in both crystal forms involving interac-
tions between transmembrane helices I and II and helix VIII is parallel and
consistent with the tridimensional model based on the paracrystalline arrangement
of rhodopsin observed in native membranes (
Fotiadis et al., 2004
). In the first
part of this chapter, we describe a protocol for the purification and crystalliza-
tion of rhodopsin in trigonal form, highlighting its most innovative step, the
(NH
4
)
2
SO
4
-induced phase separation used to concentrate purified rhodopsin prior
to crystallization.
24.1.1
Materials
All procedures are performed in a dark room under dim red light at room temperature
or colder. The room is equipped with a floor centrifuge, microfuge, spectrophotom-
eter, basic stereo microscope, and 4
C incubator. Red filters or aluminum foil is used
to cover any non-red light from the instruments. This protocol can be scaled down
about 10-fold without significant loss in the final rhodopsin yield.
24.1.1.1
Rod outer segment (ROS) isolation with sucrose gradient
1.
100-150 fresh or frozen, dark-adapted, bovine retinas
2.
Kuhn's buffer: 67 mM potassium phosphate, pH 7.0, 1 mM Mg(OAc)
2
, 0.1 mM
EDTA (ethylenediaminetetraacetic acid), 1 mM DTT (1,4-dithio-
DL
-threitol)
3.
45% sucrose in Kuhn's buffer
