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Protein Crystallization Blockcourse Structural Biology and Biophysics 2013
Badri Dubey
02.10.13
Why is it necessary to grow crystals?
Growing a suitable crystal is such a hurdle!
02 / 10 / 2013
Protein Crystallization
1. Introduction: Historical background
2. Understand how crystals grow
3. Discuss techniques for crystallizing proteins
4. Initial characterization of a crystal
Badri-Claudia-Amit 02 / 10 / 2013
Protein Crystallization
1. Introduction: Historical background
2. Understand how crystals grow
3. Discus techniques for crystallizing proteins
4. Initial characterization of a crystal
02 / 10 / 2013
Crystallisation - A short History
02 / 10 / 2013
In 1611, Kepler suggested that snow flakes derived from a regular arrangement of minute brick-like units
-> the essential idea of a crystal
SnowCrystals.com!
02 / 10 / 2013
Crystallization - A short History Hemoglobin crystals
1840, Crystallisation of earthworm hemoglobin by Hünefeld
1840-1853, crystallisation of various hemoglobin from the blood of various invertebrates and vertebrates
1935, Stanley crystallized tobacco mosaic virus and showed that it remains active even after crystallisation. Sumner, Northrop and Stanley were awarded the Nobel prize in Chemistry in 1946
Trypsin crystals (1930) Lysozyme crystals
(1939)
1925, Sumner crystallised urease
1929, Northrop crystallised gastric enzyme pepsin
1859, Crystallisation of the reserve protein of the Brazil nut.
1898, Crystallisation of horse serum albumin
Serum albumin crystals
02 / 10 / 2013
Crystallization - A short History
1910: Van Laue established the theory of diffraction of X-rays by crystals (Nobel Prize in Physics 1914) 1912: Bragg’s Law of diffraction: Diffraction is observed if X-rays scattering from a plane add in phase and this happens if the path difference 2dsinΘ is equal to nλ 1912: First structure (NaCl)
W.H. Bragg & W. L. Bragg (Nobel Prize in Physics 1915)
1895: Discovery of X-rays by Röntgen (Nobel Prize in Physics 1901)
02/ 10 / 2013
Crystallization - A short History
1949-57: D. Crowfoot-Hodgkin et al. solved the structures of penicillin (1949) and vitamin B-12 (1957). She won the Nobel Prize in Chemistry in 1964.
1953: Double helix structure of DNA
Francis Crick, James Watson and Maurice Wilkins (not in the picture)
The Nobel Prize in Physiology or Medicine 1962 Rosalind Franklin
1934: J. D. Bernal and D. Crowfoot-Hodgkin produced the first X-ray diffraction pattern of a protein: pepsin
Dorothy Hodgkin
02 / 10 / 2013
Crystallization - A short History
Protein Crystallography: One of the best techniques that reveals the atomic structure of protein, protein complexes, viruses
1971: Creation of the Protein Data Bank 1972: 2 structures 1974: 12 structures 2013: 83190 Structures were determined by X-ray crystallography
03 / 10 / 2012
Protein Crystallization – a Bottleneck
Protein crystals are more difficult to grow than mineral crystals >> properties of protein crystals and mineral crystals
Why? • high degree of mobility at the surface • low chemical and physical stability of macromolecules • need to have soluble protein in aqueous solution at high concentration
• high solvent content (30 to 80%) • soft and crush easily
• low solvent content • hard
Protein crystals vs mineral crystals
• sensitive to dehydration or change in temperature, pH or ionic strength
• small and less well ordered
• crystallisation condition are not predictable
• resistant to dehydration or change in temperature, pH or ionic strength
• highly ordered
• obtainable via temperature gradients or direct solvent removal
Protein Crystallization
02 / 10 / 2013
1. Introduction: Historical background
2. Understand how crystals grow
3. Discus techniques for crystallizing proteins
4. Initial characterization of a crystal
The process of protein crystallisation is governed by the change of Gibbs free energy. ΔGcryst has to be negative for crystal formation to be possible
02/ 10 / 2013
Protein Crystallization – Thermodynamics
ΔGcryst = ΔΗcryst −Τ ΔScryst < 0
ΔΗcryst: enthalpic contributions (heat of crystal formation) are weakly negative and derive from the side chain interactions between protein molecules within the crystals, which replace the protein-solvent interaction (Hprotein-protein - Hprotein-solvent).
ΔScryst= ΔSprotein + ΔSsolvent
ΔSprotein:: loss in rotational and translation freedom of the protein in the crystal lattice if compared with protein in solution >> negative term (-25 to -75 cal*mol-1*K-1) ΔSsolvent: gain in the solvent entropy due to the release of water molecules from both hydrophobic and polar surface residues >> positive term (5 cal*mol-1*K-1 per one water molecule released)
ΔSsolvent: gain in the solvent entropy due to the release of water molecules from both hydrophobic and polar surface residues >> positive term (5 cal*mol-1*K-1 per one water molecule released)
Nucleation: specific interactions necessary for crystal formation are established Crystal growth: ordered addition of single molecules or ordered aggregates Cessation of growth: solution is depleted of protein molecules or crystal
surfaces become covered by impurities or denatured protein
02 / 10 / 2012
Protein Crystallization – Three major steps
Precipitant concentration
02 / 10 / 2013
Visualizing the crystallization process – The Phase diagram
Principle: the release of water molecule from protein surface induces change in protein solubility which may lead either to crystallisation or to precipitation
• The release of water molecule can be achieved by adding to the protein solution molecules called precipitants (salts, ploymer, organic solvent) which compete for water molecules.
• By increasing precipitant concentration more and more, water molecules are withdrawn from the protein surface and protein molecules becomes increasingly dehydrated and start to self-associate in order to satisfy their electrostatic requirements. • The crystal growth takes place in the
metastable zone.
low moderate high
Undersaturation solubility curve
supersolubility curve
Nucleation zone
Precipitation zone
Prot
ein
conc
entr
atio
n
Precipitant concentration
Supersaturation
03 / 10 / 2012
Protein Crystallization – Ways to supersaturation
• Direct removal of the water (evaporation) • Direct mixing (batch method) • Add polymer that produces volume
exclusion • Removal of solubilizing agent • Add ligand • Change pH • Alter temperature • Alteration of the dielectric constant of the
medium (e.g. ethanole, MPD..) • Alter precipitant concentration (salts or
PEG)
02 / 10 / 2013
Precipitants used in protein crystallization
● Polymers (non-denaturing): polyethylene glycol (PEG400 to 200000), jeffamine T, polyamine)
● Salts (divalent and trivalent ions dehydrate protein more efficiently): ammonium or Na-sulphate, Li-sulphate or chloride, Na- or K-phosphate or citrate, Mg- or Ca-sulphate, Ca-chloride, Na-formate etc.
PEG
03 / 10 / 2012
Fundamental principles– Kinetic aspects
• Although thermodynamically favourable conditions are necessary, crystallization also requires suitable kinetic parameters.
• Such parameters are influenced by the path along which supersaturation is reached and by the methods used to achieve protein crystallisation.
Protein Crystallization
02 / 10 / 2013
1. Introduction: Historical background
2. Understand how crystals grow
3. Discus techniques for crystallizing proteins
4. Initial characterization of a crystal
Protein Crystallization– Methods and Techniques
Batch crystallization Vapour diffusion Micro-dialysis Liquid-liquid free interface diffusion Seeding
02 / 10 / 2013
02 / 10 / 2013
Protein Crystallisation– Microbatch
A concentrated protein solution is mixed with a concentrated solution of precipitant to produce a final supersaturated solution, which may lead to crystallization.
A: protein stays undersaturated B: protein crystallizes and the concentration of the protein in solution drops to saturation C: protein precipitates, but crystals may still grow
Protein Crystallization– Vapor diffusion
The precipitant (ppt) and protein solutions are mixed (usually 1:1) in the drop. The difference in ppt concentration between the drop and the well solution is the driving force which cause water to evaporate from the drop. Protein concentration is achieved via vapor diffusion.
[ppt]drop = [ppt]reservoir/2
H20 H20
Hanging drop Sitting drop
Protein Crystallisation– Microdialysis
The protein solution is sequestreted from the precipitant solution by a semipermeable membrane that allows the precipitant solution to slowly mix with the protein molecules (which cannot cross the membrane).
02 / 10 / 2013
Liquid-Liquid Free interface diffusion
Liquid-liquid free interface diffusion is a method of protein crystallization in which protein and precipitant gradually diffuse under the influence of a concentration gradient.
Protein sample (blue) and precipitant solutions (orange) are loaded into diffusion chambers within the chip. When interface valves open, the two solutions mix by diffusion only. This slow mixing exposes the protein to a wide swath of crystallization phase space.
Seeding
02 / 10 / 2013
Protein Crystallisation – Strategy
A multi-parametric process
Protein precipitant
concentration purity
additives
cofactors ligands
detergents
reducing agents
ageing of the sample
purity concentration
conformational heterogeneities
batch effects temperature
pH
Ionic strength
density and viscosity Volume and geometry
of samples and set-ups
Claudia Massa - Amit Sundryial 02 / 10 / 2013
02 / 10 / 2013
Protein Crystallization – Strategy
The empirical approach based on a trials and errors process (“art”)
Incomplete factorial screening Incomplete factorial screening is a method of sampling parameter
space evenly and efficiently. Factor levels are chosen
randomly and then balanced to achieve uniform sampling.
Full Factorial In full factorial screens, all elements of the matrix of parameters are sampled
Sparse matrix Sparse Matrix screens
involve an intentional bias towards combinations of
conditions that have worked previously.
02 / 10 / 2013
Protein Crystallisation – Strategy
-> identification of “Hit” conditions
Protein Crystallisation
1. Introduction: Historical background
2. Understand how crystals grow
3. Discus techniques for crystallizing proteins
4. Initial characterization of a crystal
02 / 10 / 2013
02 / 10 / 2013
Initial characterization of a crystal
02 / 10 / 2013
From the diffraction to the electron density map
1. crystal 2a. diffraction pattern 3. electron density map
& 4. fitting a
model
2b. Phases
Protein Crystallisation – References
A brief history of protein crystal growth, Alexander McPherson (1991) Journal of crystal growth 110:1-10 Introduction to protein crystallization, Alexander McPherson (2004) Methods 34: 254-265 Protein crystallisation: from purified protein to diffraction quality crystal, Naomi E. Chayen and Emmanuel Saridakis (2009) Nature Methods 5:147-153 Protein crystallization: techniques, strategies and tips, Terese M. Bergfors, International University Line (1999) P.C. Weber, Overview of protein crystallization methods, Methods in enzymology A 276 (1997)13. Mirjam Leuni, An essay on several aspects of protein crystallization research (2001). Biomolecular crystallography, Bernhard Rupp, Garland (2010), Chapter 3: Protein crystallization
02 / 10 / 2013
http://hamptonresearch.com