Nucleation and Crystal Growth

SPC1
July 2007, Hamburg
Jeroen R. Mesters
University of Lbeck, Germany
 Flowchart
Protein Solution


Supersaturation


>> Nucleation <<


>> Crystal Growth <<


3D Crystal
 Solution
Solution:
Liquid phase containing solute- and solvent-particles

Equilibrium concentration (Ce) or solubility:

The concentration of solute in a solvent at equilibrium with undissolved solute,
at a given temperature and pressure.

Saturated solution: C = Ce
Supersaturated solution: C > Ce
Undersaturated solution: C < Ce

Crystal growth does not occur unless C > Ce

 Phase Diagram

phase seperation
[protein]

precipitation
C >> Ce

C > Ce
spherulites
C < Ce labile zone
metastable zone
soluble

[precipitant]
 Nucleation
Nucleation is a phenomenon whereby a nucleus, such as a
dust particle, a tiny seed crystal, or more commonly in
protein crystallography, a small protein aggregate, starts a
crystallization process.

Nucleation can occur in the labile zone, a zone not

optimal for crystal growth!

Nucleation poses a large energy barrier, which is easier to

overcome at a higher level of supersaturation.
Free energy
Nucleation barrier energy
Surface energy contribution

G* or nucleation barrier energy
Cluster radius
Volume energy contribution
Nucleation is a process
in which the first tiny
solid aggregates are
formed. Two types of
energy govern this
process:
1) Attraction
2) Surface
 Kossel Crystal
The Kossel crystal: The growth unit displays six unsaturated bonds
located perpendicular to each face of the cube.

a =1 a=2

The cohesion of the cluster is proportional to the number de internal saturated

bonds versus the number of bonds facing towards the mother solution
 Critical size

G is proportional to the clusters volume, the
probability to disaggregate is proportional to the clusters surface.

a FS=6*a2 FA=a3
1 6 *12 13
2 6 *22 23
3 6 *32 33
4 6 *42 43
5 6 *52 53
6 6 *62 63 critical size
7 6 *72 73
8 6 *82 83
9 6 *92 93
10 6 *102 103
 Addition of precipitant
If no precipitant has been added, the
protein molecules whirl around in the solution,
described by Brownian motion. The molecules
repel each other. The system is in equilibrium.

After addition of precipitant the protein

molecules more often form small groups
of loosely linked molecules. They attract each
other. The system is no longer in equilibrium.
 Cluster formation

Without precipitant With precipitant

Time, volume and concentration
dependent process
 Common difficulties

1. If supersaturation is too high, too many nuclei form,

hence a shower of tiny crystals will appear

2. Bad nuclei may stimulate formation of amorphous

precipitate

3. In supersaturated solutions that dont experience

spontaneous nucleation, crystal growth often only occurs
in the presence of added nuclei or seeds
 Threesome

Growth

Nucleation

Concentration

But, of course, the molecules must play along!

 Solution Properties of Proteins
Physicochemical properties:
- surface properties, shape
- pI, post-translational modifications

pH:
- pH extremes fold disruption,
- if pH = pI
solubility 

Temperature:
- class I  solubility with  temp., most common
- class II little or no temperature effect
- class III  solubility with  temp.

Cosolvents:
-salts, polymers, alcohols, etc.
 Protein/Salt mixtures
The Hofmeister Series (1888)

Precipitating Power (negatively charged ovalbumin):

Anions: sulphate > phosphate > acetate > citrate > tartrate > bicarbonate
>chloride > nitrate >> chlorate > thiocyanate

Cations: Li+ > Na+ > K+ > NH4+ > Mg2+

Most stabilizing >> Most destabilizing

Salting out >> Salting in

- Negative net charge collagenese (pI 4.1, set up pH 7.2)

phosphate > sulfate > citrate > chloride
(ammonium sulfate with some sodium chloride)

- Positive net charge lysozyme (pI 9.5, set up pH 4.8)

thiocyanate > nitrate > chloride > acatate > citrate
(inversion of Hofmeister series!!)
 Electrolytes
Proteins (most proteins) are poly-electrolytes!
Protein solubility
Salting in Salting out

Inverse Salting in

[salt]
 pI and Salts

What is the pI of your protein,

and

what salt are you using and how much of it?

Lysozyme

Wrong salt?
50 to 200 mM sodium acetate!
 Protein purity/concentration
Naturally, both as high as possible but,
sufficient amount left to screen with.

If concentration via centrifugation or dialysis

(protein sticks to membrane) does not work,
immediately check for aggregation!

HIV Pr crystals on dialysis membrane

 Cessation of growth
Caused by the development of growth defects or the
approach of the solution to equilibrium.

Mother liquor
The solution in which the crystal exists - this is often
not the same as the original crystallization screening
solution, but is instead the solution that exists after
some degree of vapor diffusion, equilibration through
dialysis, or evaporation.
Phase diagram for lysozyme precipitated with 5% NaCl

Courtesy Jan Drenth

 Modern view on the nucleation
of proteins
The small unstable aggregates of the classical theory are
not solid particles but are fluctuations in the protein
concentration. Their amplitude increases gradually

Locally, high regions of protein concentration are formed.

Imagine these regions as minidroplets. If they reach a
sufficiently high concentration a real solid nucleus forms
inside that minidroplet.

Rapid nucleus formation is easy in a concentrated protein

solution. It requires overcoming the surface energy and
that is low in a concentrated solution
Phase separation curves for lysozyme
at three NaCl concentrations
Phase separation
depends on the strength
of the interaction between
the protein molecules

It is weak for 3% NaCl

and the curve is at a low
position.

It is strong for 7% NaCl;
this curve is high in the
diagram
 Fluctuations as oscillations

Fluctuations express themselves as oscillations in the phase diagram

When they reach the phase separation curve real phase separation happens in a
very tiny volume (microdroplet). Nucleation follows very soon
This can only happen below Tc
Above Tc the fluctuations do not meet the phase separation curve; they have to go
a long way before they are sufficiently large for nucleation. Nucleation above Tc is
very slow
A Lysozyme experiment
The induction time for
crystallization is plotted
as a function of the
temperature

A sharp rise happens

near the critical
temperature Tc = 22.5 oC
Optimal crystallization
conditions
Crystallize to the right
of the solubility curve
but not inside the
phase diagram curve

The best conditions

are at the level of Tc

How to determine Tc?

 Determination of an approximate value for Tc

If the induction time is extremely long one is above Tc

Action: Lower the temperature or move Tc upwards by increasing the precipitant
concentration

If crystals appear very fast one is below Tc

Action: Increase the temperature or move Tc downwards
The other phase diagram
The metastable zone
corresponds to the slow
region above Tc

The nucleation zone is the

region below Tc to the left of
the phase separation curve

The precipitation zone

corresponds to the region
inside the phase separation
curve
Supersaturation and Temperature
 Conclusions
Precipitating agents transfer the protein solution from an
unsaturated to a supersaturated state
On the road to the crystalline state an intermediate state
is formed
In this intermediate state fluctuations in the protein
concentration grow
When they reach a sufficiently high level nuclei form
Above the critical temperature Tc of liquid/liquid phase
separation this process takes a long time
Below Tc the formation of nuclei is fast
Optimal crystallization conditions are at the level of Tc
Building blocks
Growth of the crystal
Crystal morphology and size
 The END

..may the crystal force be with you..

thank you all for your patience and attention!