Biology Reference
In-Depth Information
24.2.2.2
Mesophase crystallization
24.2.2.2.1
Preparation of monoolein/cholesterol/protein mixture
To prepare a 10:1 mol/mol monoolein/cholesterol mixture, codissolve appropriate
amounts of these lipids in chloroform/methanol (2:1 v/v), evaporate the solvent with
a gentle stream of nitrogen, and then keep the dried lipid under high vacuum for at
least 6 h. Finally, seal the vials under an argon or nitrogen stream and store them at
20
C for future use.
Bring the cholesterol-doped monoolein vial up to room temperature for at least an
hour. Open the lipid vial and place it at 42
C until the solid is evenly melted. Clean
all syringes thoroughly with methanol first and then water. Then dry out the syringes
to remove water before introducing lipids. Melt pure monoolein separately and mix it
with doped monoolein if the molar monoolein/cholesterol ratio needs to be higher
than 10. With a 200
L of melted lipid/cholesterol mix-
ture through the open end of the syringe with coupler secured at the other end of a
250
m
L pipette, introduce 50-75
m
L Hamilton syringe (use of an uneven coupler for LCP mixture (
Caffrey &
Cherezov, 2009
)). Introduce the concentrated protein sample into a 100
m
L syringe
in a volume sufficient to fully hydrate the lipid (for monoolein at 20
C, full hydra-
tion with water occurs at
m
40% (w/w) water). Typically, if one is using a manual
setup, the volume of lipid should be adjusted to
22
m
L and the volume of protein
sample to 14
m
L, which will produce enough LCP mixture to set up
175 nL of
96
2 conditions for manual screening. Both syringes are coupled through an uneven
coupler and both solutions are mixed until a clear solution is obtained. Rapid disap-
pearance of the initial turbidity upon mixing indicates a good LCP sample. Overcon-
centrating the detergent during centrifugal concentration of the sample often results
in turbid LCP mixtures. Also, protein concentrations below 10 mg/mL often do not
produce crystals. The lipid/protein mixture should appear completely clear and
nonbirefringent when a drop is placed between two cover slips and observed under
crossed polarizers.
24.2.2.2.2
FRAP assay
To attain buffer and precipitant conditions conducive to diffusion, nucleation, and
crystal growth, it is advisable to subject a few micrograms of purified protein to a
fluorescence recovery after photobleaching (FRAP) assay (
Cherezov, Liu,
Griffith, Hanson, & Stevens, 2008
). Use a portion of purified material for labeling
with Cy3 mono-NHS-reactive dye (as per manufacturer's instructions) and repurify
the labeled protein on a gel filtration column to separate the free dye. Use this Cy3-
labeled protein, at 1 mg/mL, to analyze the mobility of samples in a FRAP assay by
employing a FRAP screen (
Xu, Liu, Hanson, Stevens, & Cherezov, 2011
).
Initially, a FRAP screen with 48 salts (Stock options Salt kit) combined with PEG
400 concentrations of choice (e.g., 25% in the upper 48 wells and 30% in the lower 48
wells) is set up in a single 96-well plate at pH 7.0. The same plate is then replicated
but at two other pHs (e.g., pH 6.0 and 8.0). In addition, several additives can be
individually set up to evaluate the effect of each on the protein diffusion rate.
Alternatively, one can substitute a different PEG for PEG 400 in FRAP screens.
Depending on the outcomes
from such FRAP experiments, crystallization
