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!

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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

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Crystallisation - A short History

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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!

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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

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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)

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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

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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

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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

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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

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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

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Protein Crystallization – Three major steps

Precipitant concentration

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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

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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)

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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

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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

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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

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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).

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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

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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

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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.

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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

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Initial characterization of a crystal

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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

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