Beruflich Dokumente
Kultur Dokumente
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
Saturated solution: C = Ce
Supersaturated solution: C > Ce
Undersaturated solution: C < Ce
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 is a process
in which the first tiny
solid aggregates are
G* or nucleation barrier energy
formed. Two types of
energy govern this
Cluster radius process:
1) Attraction
2) Surface
Volume energy contribution
Kossel Crystal
The Kossel crystal: The growth unit displays six unsaturated bonds
located perpendicular to each face of the cube.
a =1 a=2
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.
Growth
Nucleation
Concentration
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)
Inverse Salting in
[salt]
pI and Salts
and
Wrong salt?
50 to 200 mM sodium acetate!
Protein purity/concentration
Naturally, both as high as possible but,
sufficient amount left to screen with.
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