Biology Reference
In-Depth Information
Glucose, sucrose, paraffin oil, and several other cryoprotectants have been used
successfully with rhodopsin crystals. Most of the crystals were cryoprotected by add-
ing
3 M (NH
4
)
2
SO
4
in 50 mMMES, pH 6.35, to the crystal drops before
harvesting. The presence of buffer also slows (NH
4
)
2
SO
4
crystal formation due to
evaporation, a benefit especially if multiple rhodopsin crystals are being harvested
from the same drop.
Notes
: Rhodopsin crystals can be grown without the (NH
4
)
2
SO
4
-induced phase
separation step, and even without ROS isolation and NG/Zn(OAc)
2
extraction. Thus,
rhodopsin can be solubilized directly from retinas with NG or DDM and purified
with immobilized 1D4. This procedure, if done carefully, produces a sample of rho-
dopsin at
10
m
Lof
5 mg/mL that can be used directly for crystallization without a concen-
tration step (
Salom, Le Trong, et al., 2006
). However, crystals obtained this way
diffracted poorly and “melted” upon white light illumination, much like the original
tetragonal rhodopsin crystals (
Okada et al., 2000
). Inclusion of each additional pu-
rification step resulted in an increased quality of rhodopsin crystals, and the
(NH
4
)
2
SO
4
-induced phase separation was essential
>
for obtaining light-stable
crystals.
MERPOL DA instead of MERPOL HCS as an additive produced rhombohedral
rhodopsin crystals diffracting to 3.8
˚
(
Salom, Le Trong, et al., 2006
). However,
these crystals were more difficult to reproduce and, upon illumination, lost diffrac-
tion more dramatically than the trigonal crystals. Other additives, especially
FIGURE 24.3
Photoactivated rhodopsin crystals surrounding a colorless (NH
4
)
2
SO
4
crystal.
