Biology Reference
In-Depth Information
crystallization trials because it can be difficult to distinguish these two crystal forms
under red light.
Spin down briefly at
13,000
g
to compact the detergent/rhodopsin (det/rho)
phase.
With a wide bore 1-mL pipette tip, transfer the top, viscous det/rho phase to
microfuge tube(s) (preferably, 2 mL dolphin-nose tubes). Sometimes, the whole
det/rho phase holds together and can be transferred to the tube in one move. But
often the pipette tip fills up with the (NH
4
)
2
SO
4
phase and just a fraction of
the det/rho phase. When the microfuge tubes are full, a brief spin will condense
the det/rho phase and the bottom phase can be removed with a gel-loading tip.
This process must be repeated several times until all the det/rho phase is
transferred to the microfuge tubes and all possible (NH
4
)
2
SO
4
has been removed
from underneath. About 70% of the initial rhodopsin is recovered after this step.
Notes
: This process is slightly less efficient when rhodopsin is purified in octyl
glucoside (OG) (
Fig. 24.2
A). We tested 16 detergents commonly used for membrane
protein purification and found that only alkyl(thio)glucoside detergents could be
concentrated to a top, detergent-rich phase with saturated (NH
4
)
2
SO
4
(
Fig. 24.2
B).
Therefore, in principle, any short-chain glucoside detergent could potentially be used
to purify membrane proteins prior to their concentration by this (NH
4
)
2
SO
4
treatment.
24.1.2.5
Crystallization
The concentrated rhodopsin sample can be used directly for vapor-diffusion crystal-
lization trials, without mixing with reservoir buffer. However, due to its viscosity, the
sample is easier to handle if diluted with
1 volume of crystallization buffer, another
buffered solution, or just water. Different components, concentrations, and volumes
of the diluting aqueous solution were tested as crystallization variables and a signif-
icant percentage supported crystal growth. A sitting drop over a hanging drop format
is preferred. The higher the rhodopsin concentration,
the lower the reservoir
(NH
4
)
2
SO
4
concentration needed to obtain crystals.
Trigonal rhodopsin crystals can be observed after 1 week of incubation at 4
C
and they grow to full size (
m) in 3-4 weeks. These crystals are very stable and
retain their morphology and red color for years when kept at 4
C in the dark.
For photoactivation, a microbridge containing a drop with several crystals (or a
few crystals in mother liquor) should be transferred to a new 24-well plate with res-
ervoir buffer and the well should be sealed. Upon illumination with
100
m
>
500 nm or
white light, crystals quickly turn from red to yellow due to isomerization of 11-
cis
-retinal (a potent antagonist) into all-
trans
-retinal (
Fig. 24.3
) (rhodopsin cognate
agonist). The optimal time from the start of photoactivation until crystal freezing in
liquid nitrogen is
2 h after which diffraction quality slowly deteriorates. If left in a
drop, crystal morphology is preserved for weeks or months but eventually crystals
turn colorless.
