To crystallize proteinase K and its complex with phenylmethanesulfonyl fluoride
(PMSF), a serine protease inhibitor. You will optimize an initial set
of crystallization conditions discovered earlier by sparse matrix screening
Method: Hanging drop vapor diffusion. Full matrix
Caution: PMSF is extremely destructive to the mucous
membranes of the respiratory tract, the eyes, and skin. It may be
fatal if inhaled, swallowed or absorbed through the skin. In case of
contact, immediately flush eyes or skin with copious amounts of
water. Solutions of PMSF can be inactivated by rendering it alkaline
for several hours at room temperature.
1) 5 mg of Proteinase K (ProK) from Tritirachium album purchased
from Sigma (cat. no. P2308).
2) 5 uL of 100 mM PMSF.
3) 4M ammonium sulfate (NH4)2SO4
4) Tris buffer pH 7.0 and pH 8.0
5) Distilled water
6) Greased VDX crystallization plates, 24-wells (Hampton Research
(cat. no. HR3-140).
7) Siliconized glass cover slips (Fisherbrand Microscope Cover Glass
22x22-2 cat. no. 12-540B)
1) Work in groups of 2-3 people. Each group sets up 12
crystallization conditions for the native ProK (rows A & B of the
tray) and 12 crystallization conditions for ProK-PMSF complex (Rows C &
2) Dissolve 5 mg of ProK (supplied in 0.5 mL eppendorf tube) in 100 uL of
distilled water. Using pipet tip, stir gently to dissolve the protein. No
bubbles! The final concentration of protein should be 50 mg/mL.
3) Prepare PMSF complex by pipeting 50 uL of the 50 mg/mL ProK solution
into the tube labeled PMSF. This tube contains 5 uL of 100 mM PMSF
dissolved in isopropanol.
4) Prepare reservoir solutions:
Pipet the indicated amount of reagent to each of the 24 wells. [(NH4)2SO4]
concentration varies horizontally across a row. The pH varies
vertically down the column. The volume of each reservoir is fixed at 500
uL. Be sure to mix each reservoir thoroughly when you have finished
pipetting. You may do this by gently swirling the tray in a circular
5) Prepare drops: (a) Place a row of 6 coverslips on the top of
the tray lid, at the edge of the lid. (b)With the P-20 Pipetman,
pipet 2.5 uL of the proteinase K solution onto the middle of each cover
slip. Use a steady hand to keep the drop in the form of a nice round
bead. The coverslips have been siliconized in order to enhance bead
How to avoid blowing bubbles in the
drop: Normally, you expel liquids from the Pipetman by pushing the
plunger to the second stop. The distance between the first stop and
the second stop blows air through the tip which ensures that all the
liquid is expelled. Unfortunately, this feature also blows
bubbles in the drop you are forming on the coverslip. You can avoid blowing
bubbles by pushing the plunger to the first stop only. If you
do get a bubble in your drop, you may remove the bubble by holding the
pipet vertically and just touching the top of the drop.
Pipet 2.5 uL of reservoir A1 onto the first protein drop. The protein and
reservoir will mix by convection; there is no need to mix with the
Pipetman. Mixing just increases the likelihood of smearing the
drop. (d) Add reservoir A2 to the second drop, etc. (e) Invert
the coverslips onto their respective reservoirs. If you haven't done
this before, first try inverting a coverslip carrying a drop of
water. Keep the coverslip perpendicular to the acceleration, and
banked a little. Be careful to align the coverslips to the edges of
the plastic tray. Gently press the coverslips onto the grease seals
around the edges using the point of a pipet tip. It is best not to
use your fingers because they get greasy and smear the glass, obscuring
visibility of the crystals. Don't press directly in the center of the cover
slip. (f) Repeat steps a-e for row B. (g) Remember to use the
ProK-PMSF complex for row C and D.
6) Label your tray with your names, the date, and the protein name using
a Sharpie. Crystals
will appear in a few hours and reach full size overnight.
7) Next week: One group will collect data on the native crystal.
Another group will collect data on the PMSF complex. All others will
collect data on potential heavy atom derivatives.
Interested students are invited to return on Monday to
room BH269 for 10 minutes so that they can soak their crystals in heavy
atom solution. You can make an appointment with Mike for any time
between 11:00AM-5PM by printing your name on the sheet posted on the lab
Part Two: Preparation of Heavy Atom
Objective: Based on the results of the
native gel band shift assay select a heavy atom and soak it into crystals
1) Look for a band shift in one of the lanes containing a heavy atom.
If the heavy atom complex is cationic, then the protein will have a higher mobility
in the gel (remember, the gel is run with reverse polarity). Lanes in
which no protein appears are the result of aggretagion caused by the heavy
atom, and should not be considered further for derivatization.
2) Select the heavy atom which you are most confident caused a shift.
3) Prepare a 1:1 and 1:5 dilution of the selected heavy atom.
4) Select three drops with nice crystals from the tray of proteinase K
crystals that you grew.
5) Add 1 uL of concentrated heavy atom to drop 1.
Add 1 uL of 1:1 diluted heavy atom to drop2. Add 1 uL of 1:5 diluted
heavy atom to drop 3.