Lysozyme solution as a model system for protein crystallization

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Lysozyme solution as a model system for protein
crystallization
Seth Fraden Joshua Bloustine Viatcheslav Berejnov
Complex Fluids Group Brandeis University www.elsie
.brandeis.edu
Support NASA
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Proteomics
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Protein Crystallization
Motivation Structure implies function X-ray
diffraction provides structure 4,000 of 200,000
human proteins crystallized
Tasks DNA sequence Expression Purification Equilib
rium phase diagram Nucleation Growth
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Model System
Poly-ethylene-glycol PEG
Lysozyme
Salts
NaPhosph NaCl
NaPhosph buffer salt NaCl screening salt
Mol mass 14K, ltdiametergt 20A, pI10.5-11 Posit
ive charge at neutral pH
HOCH2 - (CH2-O-CH2) n- CH2OH
Mol mass 0.2, 0.4,1,3.35,8,12,20,35
K ltdiametergt 5-70A, Partially soluble in
aqueous salt solutions
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T-C Phase Diagram
liquid
crystal
liquidus
prism crystals
metastable
dilute - dense liquid - liquid
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Crystallization
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Model Diagram
T
Rosenbaum, Zamora, Zukoski 96
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Methods

• Cloud Point
• - Tieline
• - Light Scattering Intensity

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Cloud Point Tieline Methods
Temperature controlled microscope stage
Cloud point
initial protein solution, 1ml, Troom
capillary, lt1ul
Tie-line
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Cloud Point Tieline Paths
poor protein phase
rich protein phase
initial solution
Troom
Liquid-liquid phase transition
Tcloud
Tcool
sharp meniscus
C
Cp
Ci
Cr
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Cloud Point Curve Tieline Results
Lysozyme, NaPhosph 0.2M, NaCl 0.5M, pH6.2
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Light Scattering Intensity Measurements
laser power detector
I0
I
IIp /I0
Ip
thermostat
Tspin
Tbin
T
protein solution
photon counter
1/I
laser
Tbin
Tspin
T
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Coexistence Curve and Spinodal Line
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Cloud Point Curve Binodal, Spinodal
Cloud point curve
binodal
spinodal
Lysozyme NaPhosph 0.2M NaCl 0.5M pH6.2
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Diagram Reconstruction
one phase
T1
stable
two phases
metastable
metastable
unstable
T2
Lysozyme, NaPhosph 0.2M, NaCl 0.5M, pH6.2
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optimal crystallization conditions

• nucleation rate high at Tc

• growth rate low

small undercooling
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U(r)
r
p
Asakura Oosawa, 58
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Does B2 correlate with phase behavior?
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Conclusions

• depletion model fails for lysozyme /salt/ PEG
• B2 does not correlate with liquid phase
  boundary

• why does depletion model fail?
• (potential of mean force inadequate
  ternary phase diagram)

www.elsie.brandeis.edu