The agony and the ecstasy of protein crystallization

1
Crystallization Laboratory

• The agony and the ecstasy of protein
  crystallization
• M230D,Jan 2008

2
Goal crystallize Proteinase K and its complex
with PMSF
MAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASH PE
FEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDD
NGS GQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAAR
LQSSGVMVA VAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYG
SVLDIFGPGTSILST WIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTA
ASACRYIADTANKGDLSNIPF GTVNLLAYNNYQA

• Non-specific serine protease frequently used as a
  tool in molecular biology.
• PMSF is a suicide inhibitor. Toxic!
• Number of amino acids 280
• Molecular weight 29038.0
• Theoretical pI 8.20

Ala (A) 33 11.8 Arg (R) 12 4.3 Asn (N)
17 6.1 Asp (D) 13 4.6 Cys (C) 5
1.8 Gln (Q) 7 2.5 Glu (E) 5 1.8 Gly
(G) 33 11.8 His (H) 4 1.4 Ile (I) 11
3.9 Leu (L) 14 5.0 Lys (K) 8 2.9 Met
(M) 6 2.1 Phe (F) 6 2.1 Pro (P) 9
3.2 Ser (S) 37 13.2 Thr (T) 22 7.9 Trp
(W) 2 0.7 Tyr (Y) 17 6.1 Val (V) 19
6.8

3
Why is it necessary to grow a crystal to solve a
protein structure by X-ray diffraction ?
4
Protein crystals are ordered (periodic) arrays of
protein molecules.
One dimensional order
Two dimensional order
protein in solution.
Three dimensional order
5
Crystals are needed to amplify the diffraction
signal.
Diffraction from a crystal is strong.
Diffraction from a single molecule is weak.
6
What is the most important property of a crystal ?
7
It is the order of a crystal that ultimately
determines the quality of the structure.
Order- describes the degree of regularity (or
periodicity) in the arrangement of identical
objects.
One dimensional order
Two dimensional order
supersaturated protein solution.
Three dimensional order
DISORDERED
ORDERED
8
Order is perfect when the crystallized object
is regularly positioned and oriented in a lattice.
9
When a crystal is ordered, strong diffraction
results from constructive interference of photons.
Interference is constructive because path lengths
differ by some integral multiple of the
wavelength (nl).
detector
5
crystal
4
3
2
6
1
5
4
3
Incident X-ray
2
1
In phase
This situation is possible only because the
diffracting objects are periodic.
10
Nonregularity in orientation or position limits
the order and usefulness of a crystal.
Rotational disorder
Translational disorder
Perfect order
Disorder destroys the periodicity leading
to Streaky, weak, fuzzy, diffraction.
11
When a crystal is disordered, poor diffraction
results from destructive interference of photons.
Interference is destructive because path lengths
differ by non integral multiple of the wavelength
(nl).
detector
7
6
crystal
2
9
Incident X-rays
.
Out of phase
.
Path lengths differences are not nl because of
disorder.
12
Crystal order (and resolution) improves with
increasing number of lattice contacts

• Potassium channel (1p7b)
• 3.7 Å resolution
• Solvent content77.7

• Trypsin (1gdn)
• 0.8 Å resolution
• Solvent content36.6

13
Lattice contacts can form only where the protein
surface is rigid.
By exposing rigid surface area, you enable new
crystal forms previously unachievable.

• Eliminate floppy, mobile termini (cleave His
  tags)
• Express individual domains separately and
  crystallize separately, or
• Add a ligand that bridges the domains and locks
  them together.
• Mutate high entropy residues (Glu, Lys) to Ala.

14
Crystallization The task of coaxing protein
molecules into a crystal.
15
Is crystallization spontaneous under biological
conditions?
A lysozyme crystal Orientation and position of
molecules are locked in a 3D array High order
Solvated lysozyme monomers Random orientation
and position
16
The barriers to crystallization
Unstable nucleus

• Energy penalty
• Lose 3 degrees of freedom in orientation of
  protein molecules
• Lose 3 degrees of freedom in translation of
  protein molecules

1 crystal (lysozyme)N

• Energy reward
• Some entropy gained by freeing some surface bound
  water molecules.
• Small enthalpic gain from crystal packing
  interactions.

N soluble lysozyme molecules
DG

• Also, nucleation imposes a kinetic barrier.
• Unstable because too few molecules are assembled
  to form all lattice contacts.

nM?Mn
17
The barriers to crystallization
Unstable nucleus

• DG is decreased and the nucleation barrier
  lowered by increasing the monomer concentration
  M.
• nM?Mn
• DGDGoRTln( Mn/Mn )
• Lesson To crystallize a protein, you need to
  increase its concentration to exceed its
  solubility (by 3x). Force the monomer out of
  solution and into the crystal. Supersaturate!

N soluble lysozyme molecules
1 crystal (lysozyme)N
DG
nM?Mn
18
Methods for achieving supersaturation.

• 1) Maximize concentration of purified protein
• Centricon-centrifugal force
• Amicon-pressure
• Vacuum dialysis
• Dialysis against high molecular weight PEG
• Ion exchange.
• Slow! Avoid precipitation. Co-solvent or low salt
  to maintain native state.
• We are going to dissolve lyophilized protein in a
  small volume of water.

Concentrate protein
19
Methods for achieving supersaturation.

• 2) Add a precipitating agent
• Polyethylene glycol
• PEG 8000
• PEG 4000
• High salt concentration
• (NH4)2SO4
• NaH2PO4/Na2HPO4Polyethylene glycol
• Small organics
• ethanol
• Methylpentanediol (MPD)

PEG Polymer of ethylene glycol
Precipitating agents monopolize water molecules,
driving proteins to neutralize their surface
charges by interacting with one another.
20
Systematic vs. Shotgun Screening

• Shotgun- for finding initial conditions, samples
  different preciptating agents, pHs, salts.
• Systematic-for optimizing crystallization
  condtions.

First commercially Available crystallization Scree
ning kit. Hampton Crystal Screen 1
21
Methods for achieving supersaturation.
Drop ½ protein ½ reservoir

• 3) Further dehydrate the protein solution
• Hanging drop vapor diffusion
• Sitting drop vapor diffusion
• Dialysis
• Liquid-liquid interface diffusion

2M ammonium sulfate
Note Ammonium sulfate concentration is 2M in
reservoir and only 1M in the drop. With time,
water will vaporize from the drop and condense in
the reservoir in order to balance the salt
concentration.SUPERSATURATION is achieved!
22
The details of the method.
23
Practical Considerations

• Begin with reservoirs
• pipet reqd amount of ammonium sulfate to each
  well.
• Pipet reqd Tris buffers, to each well
• Same with water.
• Then swirl tray gently to mix.
• When reservoirs are ready, lay 6 coverslips on
  the tray lid,
• then pipet protein drops on slips and invert over
  reservoir.
• Only 6 at a time, or else dry out.

Linbro or VDX plate
24
Proper use of the pipetor.
25
Which pipetor would you use for delivering 320 uL
of liquid?
P20
P200
P1000
26
Each pipetor has a different range of accuracy
P20
P200
P1000
200-1000uL
20-200uL
1-20uL
27
Which pipetor would you use for delivering 170 uL
of ammonium sulfate?
P20
P200
28
How much volume will this pipetor deliver?
0 2 7

29
How much volume will this pipetor deliver?
1 7 0

30
How much volume will this pipetor deliver?
0 2 7

31
What is wrong with this picture?
0 2 7

- - - -
50 mL
32
What is wrong with this picture?
- - - -
50 mL
33
Dip tip in stock solution, just under the surface.
- - - -
50 mL
34
Withdrawing and Dispensing Liquid.3 different
positions
Start position
First stop
Second stop
0 2 7
0 2 7
0 2 7



35
Withdrawing solution set volume, then push
plunger to first stop to push air out of the tip.
Start position
First stop
Second stop
0 2 7

- - - -
50 mL
36
Dip tip below surface of solution. Then release
plunger gently to withdraw solution
Start position
First stop
Second stop
0 2 7

37
To expel solution, push to second stop.
Start position
First stop
Second stop
0 2 7

38
When dispensing protein, just push to first
stop.Bubbles mean troubles.
Start position
First stop
Second stop
0 2 7

39
Hanging drop vapor diffusionstep two
Pipet 2.5 uL of concentrated protein (50 mg/mL)
onto a siliconized glass coverslip. Pipet 2.5 uL
of the reservoir solution onto the protein
drop 2M ammonium sulfate 0.1M buffer
BUBBLES MEAN TROUBLES Expel to 1st stop, not 2nd
stop!
40
Hanging drop vapor diffusionstep three

• Invert cover slip over reservoir quickly
  deliberately.
• Dont hesitate when coverslip on its side or else
  drop will roll off cover slip.
• Dont get fingerprints on coverslip they obscure
  your view of the crystal under the microscope.

41
Dissolving Proteinase K powder

• Mix gently
• Pipet up and down 5 times
• Stir with pipet tip gently
• Excessive mixing leads to xtal showers
• No bubbles

5.25 mg ProK powder
100 uL water
4 uL of 0.1M PMSF
50 mg/mL ProK
42
Dissolving Proteinase K powder

• Mix gently
• Pipet up and down 5 times
• Stir with pipet tip gently
• Excessive mixing leads to xtal showers
• No bubbles

Remove 50 uL Add to 5 uL of 100 mM PMSF
50 mg/mL ProK
55 uL of 50 mg/mL ProKPMSF complex
43
Proteinase K time lapse photography

• Covers first 5 hours of crystal growth in 20
  minute increments

500 mm
44
(No Transcript)
45
Heavy Atom Gel Shift Assay.Why?
46
Why are heavy atoms used to solve the phase
problem?

• Phase problem was first solved in 1960. Kendrew
  Perutz soaked heavy atoms into a hemoglobin
  crystal, just as we are doing today. (isomorphous
  replacement).
• Heavy atoms are useful because they are electron
  dense. Bottom of periodic table.
• High electron density is useful because X-rays
  are diffracted from electrons.
• When the heavy atom is bound to discrete sites in
  a protein crystal (a derivative), it alters the
  X-ray diffraction pattern slightly.
• Comparing diffraction patterns from native and
  derivative data sets gives phase information.

47
Why do heavy atoms have to be screened?

• To affect the diffraction pattern, heavy atom
  binding must be specific
• Must bind the same site (e.g. Cys 134) on every
  protein molecule throughout the crystal.
• Non specific binding does not help.
• Specific binding often requires specific side
  chains (e.g. Cys, His, Asp, Glu) and geometry.
• It is not possible to determine whether a heavy
  atom will bind to a protein given only its amino
  acid composition.

48
Before 2000, trial error was the primary method
of heavy atom screening

• Pick a heavy atom compound
• hundreds to chose from
• Soak a crystal
• Most of the time the heavy atom will crack the
  crystal.
• If crystal cracks, try lower concentration or
  soak for less time.
• Surviving crystal are sent for data collection.
• Collect a data set
• Compare diffraction intensities between native
  and potential derivative.
• Enormously wasteful of time and resources.
  Crystals are expensive to make.

How many crystallization plates does it take to
find a decent heavy atom derivative?
49
Heavy Atom Gel Shift Assay

• Specific binding affects mobility in native gel.
• Compare mobility of protein in presence and
  absence of heavy atom.
• Heavy atoms which produce a gel shift are good
  candidates for crystal soaking
• Collect data on soaked crystals and compare with
  native.
• Assay performed on soluble protein, not crystal.

None Hg Au Pt Pb Sm
50
Procedures

• Just incubate protein with heavy atom for a
  minute.
• Pipet 3 uL of protein on parafilm covered plate.
• Pipet 1 uL of heavy atom (100 mM) as specified.
• Give plate to me to load on gel.
• Run on a native gel
• We use PhastSystem
• Reverse Polarity electrode
• Room BH269 (Yeates Lab)