Crystallizing proteins

Overview of protein structure determination: X-ray crystallography

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Definition of study objective

Comprehensive literature search and bioinformatics

Obtain DNA and clone into vector

Express and purify soluble proteins

Initial crystallization and optimization

Harvest and flash-cool, collect data

No usable diffraction data

No interpretable map

New data, crystal, or protein construct

Density averaging and modification

Heavy atom substructure

Experimental phasing

Anomalous or derivative data

No soluble or folded protein

New protein construct, ortholog

New protein construct, tag, etc.

No diffracting crystals

Native single wavelength data

Molecular replacement

Analysis of structure, fold family, annotation, binding sites, docking

studies

Automated modeling building

Restrained maximum likelihood refinement

Validation, model correction and polishing

Model deposition

The history of (recorded) protein crystal growth started about 160 years ago. The first

published observation of the crystallization of a protein appears to be by Hünefeld in 1840 of

the protein hemoglobin from the earthworm.

This observation clearly stated that protein crystals can be produced by the controlled

evaporation of a concentrated protein solution, that is, protein crystals can be produced by

slow dehydration.

History of Protein crystallization

For the next 15 years, most of the crystals obtained from the

blood of several animals were found to be by chance. The first

person to actually devise successful and reproducible methods

for the growth of hemoglobin crystals was Fünke (1851).

For his discovery that

enzymes can be crystallized.

For his preparation of enzymes

and virus proteins in a pure form.

Nobel Prize in Chemistry (1946)

The first enzyme (urease) was crystallized by James Sumner in 1926, followed by the

crystallization of pepsin in 1930 by John Northrop.

Do you think, proteins can be purified by growing crystals?

Principles of Protein Crystallization

The methods employed in crystal production rely on the ordered precipitation of proteins.

A practical way to represent the change of protein solubility with precipitant is the solubility

diagram.

Crystallization diagrams

Obtaining suitable single crystals is the least understood step in the X-ray structural analysis

of a protein.

Protein crystallization is mainly a trial-and-error procedure in which the protein is slowly

precipitated from its solution.

Protein crystallization is an art, than science.

What are the important factors which can affect the formation of protein crystals?

As a general rule, the purer the protein, the better the chances to grow crystals. A reasonable

single-band appearance in a well loaded SDS gel (<95% purity) is certainly a good starting

point.

The purity requirements of the protein crystallographer are different and more stringent than

the requirements of the biochemist. For protein crystallization, all molecules of the protein

should have the same surface properties, especially the same charge distribution on their

surface.

Mass spectrometry is a valuable tool in protein crystallization in checking the purity of a

preparation.

Purity of the protein

Freshness and conformational state

For most proteins, degradation occurs over time, sometimes rapidly

and using the fresh protein seems to be of advantage for

crystallization. Even small amounts of degraded protein or

oligomeric aggregates may drastically hamper crystallization.

Generally, protein solution contains all kind of foreign and

endogenous detritus such as remnants from chromatography resins,

dirt, denatured and aggregated protein and other particulates.

These may well act in an uncontrolled fashion as nucleation sites

and it is thus good practice to spin the protein stock down before

aspirating the protein solution.

This is particularly advisable if the protein stock has been frozen

and thawed, where partial denaturation often occurs.

Batch variation and contaminants

It is quite common that different batches of the same protein do

not show the same crystallization behavior.

Thus, a second batch prepared from the same construct may

actually crystallize if the first one did not.

Proteins also tend to acquire all kinds of hitch hikers such as

cofactors, detergents, lipids or membrane components that co-

purify and vary from batch to batch.

Ligand binding sites in particular can attract all kind of detritus

from the environment.

Protein concentration

The often quoted rule of “at least 10 mg/ml” is not sustainable in view of the evidence.

Although the average protein concentration extracted from PDB data is around 14 mg/ml,

there are many examples of successful crystallization in the low mg range and even lower.

The required concentration depends on the individual protein and instead of an absolute value,

a more rationally defensible guideline is “as high as reasonably achievable” in each respective

case.

A majority of clear drops observed in the crystallization trials thus indicates too low a

concentration.

A few initial trials of observing a sub-μl drop of protein solution mixed with highly

concentrated precipitants such as 30% PEG 5000, 4 M ammonium sulfate or 30% isopropanol

can quickly determine whether precipitation can be achieved.

Buffers, salts and additives in protein stock

Generally, a buffer solution and a low salt concentrations may be necessary for stability the

protein. For example, weak, preferably organic buffers such as 10 mM HEPES are commonly

used.

Additives, ligands, specific cofactors or even detergents may be needed to keep the protein

stable and active and may place additional restraints on the choice of crystallization reagents.

Certain cocktail components such as Ca2+ ions and phosphate stock buffer – a favorite of

protein biochemists but less suitable for crystallization, are incompatible.

It is also rather wasteful to screen protein that is unstable below physiological pH against a

screening kit that contains a large number of low pH cocktails.

Effect of pH on protein solubility

The pH of the solution exerts a very strong effect on protein crystallization. Although the

solubility minima correspond well with isoelectric point (pI), the correlation of pI and the

actual pH of crystallization is weak, meaning that protein do not crystallize best most

frequently a their pI. The pH change is nevertheless a key parameter and immensely useful for

protein crystallization screening.

ΔpH = pI - pH

Crystal packing effects, artifacts and solvent

Despite the fact that the core structure and even the enzymatic function protein are maintained

in crystals, flexible and dynamic regions can be fixed in a specific conformation because of

crystal packing interactions and altered conformations of flexible regions may be induced.

Protein crystals contain on average around 50% solvent, mostly disordered in large solvent

channels between the stacked molecules or along plain rotation axes in the crystal structure.

The solvent contains water and all other molecules and ions present in the crystallization

cocktail, plus anything carried through from purification into the protein stock solution.

As a consequence, such an apparently specific conformation observed in a crystals structure

may not actually be a dominant representation of the that part of the protein structure in

solution.

A simple safeguard against misinterpretation, which usually implies assignment of certain

biological relevance that is de facto not warranted, is to display all neighboring molecules in

the crystal structure and examine contact regions carefully for conformations that likely a

result of crystal packing.

Determining the structure from multiple different crystal forms may also help to resolve the

question of crystallization artifacts in the structure model.

Crystal forms and morphology

It is not uncommon to observe different crystal forms under varying crystallization conditions,

and multiple crystal forms may even be present in the same crystallization drop.

This polymorphism can be used to advantage, because different crystal form may exhibit

significantly different diffraction quality.

It is worthwhile trying to optimize all the crystal forms present rather than just focusing on the

one that looks best by visual assessment in the initial screens, in part because polymorphism

can also resolve question regarding crystallization artifacts.

Effect of temperature on protein solubility

Protein solubility can either increase of decrease with temperature, often varying between

precipitants even for the same protein.

Statistics show that most protein are crystallized either at room temperature or at 4 C. This

binary choice results from the fact that traditionally protein are prepared and purified at

reduced temperature, commonly in a 4 C cold-room to slow down degradation by proteases.

Exercise: Make a histogram of crystallization temperature of the structures submitted in

the protein data bank.

Crystallization Techniques

1. Batch Crystallization

2. Vapor-diffusion method

3. Dialysis

4. Free-interface diffusion

Batch crystallization

The principle is that the precipitating reagent is instantaneously added to a protein solution,

suddenly bringing the solution to a state of high supersaturation. In this method, protein

crystals are grown by adding 1–2 μl drops containing the protein and the precipitant (1:1

ratio). The drops are suspended in an oil (e.g., paraffin oil and silicon oil). The oil acts as a

sealant to prevent evaporation. It does not interfere with the common precipitants, but it does

interfere with organic compounds that dissolve in the oil.

Vapor-diffusion: Hanging-drop method

In this method, drops are prepared on a siliconized microscope glass cover slip by mixing 3–

10 μl of protein solution with the same volume of precipitant solution. The slip is placed

upside down over a depression in a tray. The depression is partly filled with the required

precipitant solution (∼1 ml). The chamber is sealed by applying oil or grease to the

circumference of the depression before the cover slip is put into place.

Vapor-diffusion: Sitting-drop method

If the protein solution has a low surface tension, it tends to spread out over the cover slip in

the hanging drop method. In such cases, the sitting drop method is preferable.

Dialysis method

The advantage of dialysis is that the precipitating solution can be easily changed. For

moderate amounts of protein solution (more than 0.1 ml), dialysis tubes can be used. The

dialysis membrane is attached to a tube by means of a rubber ring. The membrane should be

rinsed extensively with water before use or, preferably, boiled in water for about 10 min. For a

μl amount of protein solution, one can use either a thickwalled microcapillary. The

disadvantage of the button is that a protein crystal in the button cannot be observed with a

polarizing microscope.

Free-interface diffusion method

In this method, the protein solution and the solution containing the precipitant are layered on

top of each other in a small-bore capillary. The lower layer is the solution with higher density

(e.g., a concentrated ammonium sulfate or PEG solution). If an organic solvent such as MPD

is used as precipitant, it forms the upper layer. For a 1:1 mixture, the concentration of the

precipitant should be two times its desired final concentration. The two solutions (∼5 μl of

each) are introduced into the capillary with a syringe needle, beginning with the lower one.

Spinning in a simple swing out centrifuge removes air bubbles. The upper layer is added and a

sharp boundary is formed between the two layers. They gradually diffuse into each other.

Atomic Force Microscope of crystal growth

Analyzing the outcome of crystallization trials

Do protein dislike crystallizing?

Soluble proteins in cellular compartments or intercellular space do not float around freely, but,

just like in crystals, share with other proteins a very crowded environment, full of small

molecules, nutrients, and copies of themselves and other proteins.

It is conceivable that proteins perhaps had to evolved precisely to not aggregate and associate

with each other under normal circumstances. Uncontrolled spontaneous crystallization

certainly would compromise the viability of a normal cell, and some empirical evidence

points toward the possibility of negative evolutionary design.

An interesting curiosity in this context is the fact that Bacillus thuringiensis, used

commercially a biopesticide, actually stores its insecticidal proteins as perfectly diffracting

protein microcrystals.

Crystallization of lysozyme

The most convenient protein to start with is hen egg white lysozyme. It can be obtained

commercially in pure form, is relatively inexpensive and can be used immediately for a

crystallization experiment.

Crystallization condition 1:

Lysozyme: 50 mg/ml in 0.1 M Sodium Acetate pH 4.6

Reagent: 8% w/v Sodium Chloride, 0.1 M Sodium Acetate pH 4.6

Mix equal amounts of lysozyme with reagent, incubate at 4 or 22 degrees Celsius. Batch or

vapor diffusion works fine.

Crystallization condition 2:

Lysozyme: 50 mg/ml in 0.1 M Sodium Acetate pH 4.6

Reagent: 10% v/v Ethylammonium nitrate

Mix equal amounts of lysozyme with reagent, incubate at 4 or 22 degrees Celsius. Batch or

vapor diffusion works fine.

Crystallization condition 3:

Lysozyme: 50 mg/ml in 0.1 M Sodium Acetate pH 4.6

Reagent: 2.5 M Sodium Chloride

Mix equal amounts of lysozyme with reagent, incubate at 4 or 22 degrees Celsius. Batch or

vapor diffusion works fine.