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ROBA GETACHEW DEJENE

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INTRODUCTION AND THEORY OF

CRYSTALLIZATOIN

The first step in the determination of an X-ray crystal

structure of proteins, is the growth of sufficiently

large protein crystals.

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WHAT IS PROTEIN CRYSTALLIZATION?

Is the formation of solid crystals from a homogenoussolution.

In order to arrive at a crystalline form starting from adissolved form, the solubility of the protein moleculesmust be reduced.

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CONT’D

reducing the solubility ₌ amorphous protein precipitate.

The process of crystallization can be differentiated into two steps:

A. The nucleation process, and

B. Crystal growth.

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Fig 1.Phase diagram applying to crystal growth

Cont’d

crystallization requires the formation of a supersaturated protein-precipitant solution.

The transition from a stable solution to a supersaturated solution can be achieved by increasing either;

A. The concentration of the protein or

B. The precipitant

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CONT’D

Protein crystallization occurs when the concentrationof protein in solution is greater than its limit ofsolubility.

The physical process of causing a change inconcentration can be carried out through a commonmethod called hanging drop, which utilizes a processcalled vapor diffusion.

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HOW CONCENTRATION IS INCREASED?

The concentration is increased due to vapor leavinghanging drop to reach equilibrium with its surroundings.

The most common setup to grow protein crystals is by thehanging drop technique:

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Fig.2The Hanging-Drop Set Up

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Cont’d

The contents of the hanging drop includes;

A .sample protein.

B. well solution (reservoir solution) and

C. sometimes water.

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Fig3.Hanging Drop Protocol

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PROTOCOLS OF HANGING DROP METHOD

1.The well - 1ml of a buffered precipitant solution .

2.Then 1 µl of the concentrated protein sample is pipetteonto a siliconized cover slip, followed by 1 µl of the wellsolution(reservoir solution)

3. The cover slip is then inverted over the well and sealed.

4.This is then left undisturbed for at least 24 hours

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Which Materials AreUseful As Precipitants?

Salts,

Organic polymers,

Alcohols

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Cont’d

As the protein/precipitant mixture in the drop is lessconcentrated than the reservoir solution waterevaporates from the drop into the reservoir.

the concentration of both protein and precipitantin the drop slowly increases, and crystals may form.

Crystallization----- over the course of days or weeks

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Fig 4.Cartoon of a Hypothetical Crystalline Lattice

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Applications of Crystallized Proteins

Crystallization methods has many practical applications.

determination of three dimensional structure ofproteins;

Pharmaceutical drugs

Protein engineering.

Synthetic vaccines.

proteins interaction with other molecules,

conformational changes, and

Protein catalysis in the case of enzymes16

Applications of CrystallizedProteins

Fig.4 Three-dimensional structure of pepsin. 17

Advantages and disadvantages

Advantages

the least expensive

possibility of affecting the equilibration

rate and thus approaching super saturation more slowly

ability to alter the concentration of the components in the crystallizing solution during the experiment .

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Cont’d

Disadvantages

both nucleation and rapid crystal growth can occur

simultaneously.

Investigator as only option to guess at which conditions crystallization exactly occurs.

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REFERENCEs

1. Biochemistry/ Jeremy M.Berg, John L.Tymoczko, Lubert stryer.-6th ed.

2. Jena Bioscience GmbH | Löbstedter. http://www.jenabioscience.com

3. http://www.ruppweb.org/crystal_lab/cystalmake.html

4.http://en.wikipedia.org/wiki/X-ray crystallography-Crystallization

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Merci beaucoup!!