Biology Reference
In-Depth Information
amphiphiles, had negative and positive effects on crystal and diffraction quality.
However, no other crystal form was identified from these crystallization trials.
24.2
CRYSTALLIZATION OF
b
2-AR
Here, we describe the crystallization in a monoolein/cholesterol lipidic cubic phase
(LCP) of a thermostabilized
2-AR(E122W)/T4L construct described in Hanson
et al. (
Alexandrov, Mileni, Chien, Hanson, & Stevens, 2008
; PBD ID 3D4S) as
bound to carazolol instead of timolol, with some modifications to the protocol that
made crystal growth more reproducible.
The LCP method, first used to crystallize bacteriorhodopsin (
Landau &
Rosenbusch, 1996
), already has been employed to obtain one-tenth of membrane pro-
tein structures in the Protein Data Bank (
Aherne, Lyons, & Caffrey, 2012
) and most
structures of engineered GPCRs (
Caffrey, Li, & Dukkipati, 2012
). Although no strat-
egy was found to favor a particular relative orientation of GPCRs, four of these recep-
tors crystallized in LCP appeared as dimers in a parallel arrangement (
b
b
2-AR, CXCR4,
and
-opioid receptors) (
Cherezov & Caffrey, 2007; Chun et al., 2012
).
Some of the advantages found with crystallization of GPCRs in mesophase are (i)
rapid crystal growth, (ii) mild temperatures used for crystal growth (
k
-and
m
20
C), and
(iii) the fact that all crystals obtained so far are type I, formed by stacked layers
of two-dimensional crystals that mimic the native membrane. Another strategy to
facilitate GPCR crystallization has been to modify their sequences to (i) stabilize
the receptors, (ii) remove flexible regions, (iii) enhance their expression, and
(iv) increase the receptors' hydrophilic areas. Such sequence modifications include
truncation of the third intracellular loop or its substitution by T4L or BRIL (thermo-
stabilized apocytochrome
b
562
), N-terminal fusion of T4L or BRIL, truncation of
long N- and C-termini, and/or addition of thermo-stabilizing mutations.
24.2.1
Materials
24.2.1.1
Solubilization of membranes
1.
DDM
2.
Cholesterol hemisuccinate (CHS)
3.
Dounce homogenizer, 100 mL
4.
Insect cell (Sf9) pellets expressing the receptor
5.
1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5
6.
1 M NaCl
7.
1 M MgCl
2
8.
1 M KCl
9.
Protease inhibitor cocktail, EDTA-free (Roche)
10.
Glycopeptide
N
-glycosidase (PNGase F)
11.
DNase (for example, Benzonase
®
nuclease)
12.
Iodoacetamide
