3.蛋白质结晶学-可溶性蛋白-part 1

Crystal
Solution
Concentration gradient
Flow of tranport towards the crystal face
Flow of growth units
<<<<
Flow of integration in the crystal surface
a
Crystallization taking place in a diffusive environment yields crystals of the highest quality
Crystal -> Diffraction pattern -> Electron density -> Model
Part I
• Fundamentals of macromolecular crystallization • Crystallization techniques
• Practical considerations
Classical protein crystallization techniques
Macromolecule concentration
Vapor diffusFra Baidu bibliotekon Hanging/Sitting drop
20 18 16 14 12 10 8 6 4 2 0 0 5 10 15 20 25 30 35 40 45 50
Counter diffusion crystallization
Raphael Eduard Liesegang (1869-1947)
4Ag+ + Cr2O7-2 + H2O -----> 2Ag2CrO4 + 2H+
History of a supersaturation wave Counter-diffusion technique How it works?
Prerequisites for protein crystallisation. • Need about 10 mg purified protein. - Various forms of chromatography. - Better than 95 % purity if possible. • Must be homogeneous. - Protein isoforms & microhetrogenity very damaging to crystal growth. • Typically concentrate to about 20 mg/ml. • Must be stable throughout the experiment. - Can take days, weeks or months to grow crystals.
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Crystallization trial will be under kinetic control when the relative supersaturation, (t ) , defined by
16 14 12 10 8 6 4 2 0 0 5 10 15 20 25 30 35 40 45 50
E' E 0' 0
Understurated zone
Labile zone
Metas table zone
Precipitating agent concentration
Typical purification protocols. • Grow cells (E. coli, yeast etc). • Break cells (French press or sonication or lysozyme). • Separate eg. membranes from other things by centrifugation. - Extract supernatent, resolubilise membrane proteins or inclusion bodies. • Purification: - Ion exchange chromotography; affinity chromotography (His tag); Gel filtration most common. Isoelectric focussing a less common option. • Check purity on a SDS gel. - Other biophysical characterisation such as activity assays, dynamic light scattering, etc. • Frequently change buffer. • Concentrate to typically around 10 to 20 mg/mL. • Crystallisation setups.
Part 2
• Advanced techniques: Crystallization screening with Trace-Fluorescent labeling
Part 3
• Growing large crystals for neutron crystallography • Space
Protein crystallization for x-ray and neutron crystallography
Joseph D. Ng
iXpressGenes Inc University of Alabama in Huntsville
Ixpressgenes.com Nglab.uah.edu
Factors affecting protein solubility. • pH - As pH changes certain groups (eg. Asp, Glu, Lys, His, Arg, Try) go from neutral to charged, or from charged to netural. - Alters surface charges & interactions with water. • Salts affect protein surface charges & interact with water. - Salting in (adding salt increases protein solubility). - Salting out (adding salt decreases protein solubility). • Polar solvents. - eg. polyetheleneglycol (PEG) soaks up water. • Temperature - Thermodynamic factors influence solubility.
Nail Polish
Precipitant + glycerol (cryo solution) + derivative
Nail Polish
Volume = 5-7 ul
Protein and low concentration agarose
Counter-diffusion technique
• • • • Super(under)saturation is immediately achieved Screening performed by repeating a number N of experiments using different initial conditions Higher the number N of experiments higher the possibility of success Number of visited crystallization conditions per trial: One supersaturation value, One (infinite) rate of supersaturation
•
Classical protein crystallization techniques: Batch method
20
Macromolecule concentration
Direct mixing of protein and precipitant
18 16 14 12 10 8 6 4
a) Batch experiment
E
Labile zone
Metas table
Understurated zone
Oil thin layer
zone
Oil
2 0
0
0 5 10 15 20 25 30 35 40 45 50
Protein + Precipitant
Precipitating agent concentration
110 101
Growth rate
Supersaturation
b
Dettachment Attachment
c
d
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Fast flow of P.A. molecules Sl ow flow of protein molecu les
P.A. chamber
Protein chamber x
L
a)
0
Time = 0
Buffe r chamber
P.A. chamber
Protein chamber
0
x
b)
L
Time = 0
c(t ) (t ) 1 c s
(t ) (t )
< 0
> 0
Thermodynamic Control
Kinetic Control
NUCLEATION
20 18
b) Vapour diffusion experiment
Macromolecule concentration
The micro-batch method is well suited for screening including miniaturization and automation. It is fast to implement and minute drops can be used.
Trial is blind
b) Vapour diffusion experiment
E' E 0' 0
Understurated zone
Labile zone
Metas table zone
Transport of water molecules
Precipitating agent concentration
Protein solution + precipitant at low concentration Evaporation of water increases the concentration of precipitating agent and protein at the same rate. Precipitant at high concentration (usually twice the one in the drop) • Each experiment explores few supersaturation values at constant rate of change of supersaturation. Higher the number N of experiments higher the possibility of success