Crystallization

Four major steps in crystallizationObtain large amounts of pure protein

samplesChoose a protein buffer in which the

protein is both soluble and stableBring protein solution to supersaturation

where spontaneous nucleation can take place

SolubilityAs a rule, protein solubility will usually increase as you add salt to your aqueous solution, then begin to decrease when the salt concentration gets high enough to compete with the protein for

hydration (interaction with water molecules).

Diagram from the website of Alan Clark, Victoria University of Wellington, New Zealandhttp://www2.vuw.ac.nz/staff/alan_clark/teaching/index.htm

HbCO (carboxyhemoglobin) solubility as a function of ionic strength in the presence of several different types of salts

Nucleation

A phenomenon whereby a “nucleus”, such as a dust particle, a tiny seed crystal, or commonly in protein crystallography, a

small protein aggregate, starts a crystallization process.

Nucleation poses a large energy barrier, which is easier to overcome at a higher level of supersaturation.

Common difficulties:

1. If supersaturation is too high, too many nuclei form, hencean overabundance of tiny crystals.

2. In supersaturated solutions that don’t experience spontaneous nucleation, crystal growth often only occurs in the presence of added nuclei or “seeds”.

Cessation of growth

Caused by the development of growth defects or the approach of the solution to equilibrium.

Mother liquor

The solution in which the crystal exists - this is often not the same as the original crystallization screening solution, but is instead the solution that exists after

some degree of vapor diffusion, equilibration through dialysis, or evaporation.

Major factors that affect crystallization

1) Purity of proteins

2) Protein concentration

3) Starting conditions (make-up of the protein solution)

4) Precipitating agent (precipitant)

5) Temperature

6) pH

7) Additives: Detergents, reducing agents, substrates, co-factors, etc.

1) Purity of proteins

Sources of heterogeneity (other than unrelated proteins and nucleic acids as contaminants):

• Partial proteolysis products• Oxidation of cysteines• Deamidation of Asn and Gln to Asp and Glu• Post-translational modifications• Oligomerization• Isoforms• Misfolded population• Structural flexibility

2) Protein concentration

Consistency and reproducibility are the major issues with protein concentration a reliable assay for determining the concentration.

• Bradford Assay (BSA is used as a standard)

E. Coli expression systems are crystallographers’ most commonly used method of obtaining protein. Problems can arise from low expression yields:

• Cytotoxic - your protein is killing your E. coli• Unstable plasmid or mRNA • Protein is misfolded (coexpress with GroEL?)• Some common eukaryotic codons are rare in E. coli

3) Starting conditions (make-up of the protein solution)

The main point is to KNOW what your starting conditions are for purposes of reproducibility.

4) Precipitating agent (precipitant)

SaltsAmmonium sulfateSodium chloridePotassium phosphate

Organic reagentsMPDIsopropanol

Polyethylene glycolPEG 4000PEG 6000PEG 8000

Comparison of Crystallization Comparison of Crystallization and Precipitationand Precipitation

Description Crystallization PrecipitationSolubility Wide range, usually

medium to highSparingly soluble

Relative supersaturation

Low High

Product morphology Well-defined Ill-definedProduct crystal size Large SmallNucleation mechanism

Secondary Primary

Nucleation rate Low HighGrowth Rate Wide Range LowControllability Controllable Difficult to control

5) Temperature

Temperature affects protein stability and also the dynamics of

how protein solution reaching supersaturated states. Ideally:

• An individual crystal screen should be kept at constant temperature

• Each set of conditions should be screened at several temperatures

• The easiest are 4 C and room temperature, also try 12 or 15 C

6) pH

Surface charges affect “crystal packing”. (Crystal packing refers to the spatial arrangement of molecules within the crystal, particularly in reference to their relationships to one another.)Hydrophobic interactions are less important than electrostatic interactions in crystal packing.

7) Additives:

Sometimes you can increase the stability of your protein,and/or the homogeneity of its conformation by having relevant additives present in the crystal screen:

• Detergents • Reducing agents• Substrates• Co-factors• etc.

Still no crystals after thorough screening. Now what?

New constructs Deletion mutants

Complexes with substratesProtein complex with Fab fragments

Homologous proteins

Fab

Crystallization of membrane proteinsThe lipidic cubic phase method (Landau and

Rosenbusch)

Cocrystallization with Fab fragments

Common Methods for Crystallization:

•Vapor Diffusion •Slow Evaporation•Dialysis

Hanging Drop Vapor Diffusion

Most popular method among protein crystallographers.

1. Crystal screen buffer is the well solution (0.5 - 1 mL)2. Drop (on siliconized glass cover slip) is 1/2 protein solution, 1/2 crystal screen buffer (6-10 L). So, the concentration of precipitant in the drop is 1/2 the concentration in the well.3. Cover slip is inverted over the top of the well and sealedwith vacuum grease (airtight).

4. The precipitant concentration in the drop will equilibrate with the precipitant concentration in the well via vapor diffusion.

Sitting Drop Vapor Diffusion

Same basic principles as in hanging drop method, except the dropcontaining your samplesits on a bridge within the well. This allows fora larger sample size (20 -40 L), however protein is frequently precious to the crystallographer, so there isn’t that much demand for a larger samplesize.

Oil Immersion Micro Batch

This method is rising rapidly in popularity- typical sample size 1-6 L

Figure 1- Paraffin oil allows for little to no diffusion of water through the oil. This is a true batch experiment because all the reagents are present at a specific and relatively unchanging concentration.

Figure 2- Al’s oil is a 1:1 mixture of silicon oil and paraffin oil which allows for evaporation through slow diffusion through the oil. This is an evaporation Method, and the concentration of the protein and reagents in the drop does increase over time.

Microdialysis

Dialysis buttons can be purchased for a wide range ofsample sizes (~ 5 - 350 L).

In the dialysis experiment, the sample is often introduced to high salt concentrations within the button that are allowed to equilibrate with lower salt concentrations in the buffer over time. This is known as a “salting-in” method. It exploits the fact that not only does protein solubility tend to decrease with very high ionic strengths, it also has a minimum at very low ionic strength.

NucleationThe generation of ultramicroscopic particles in the process of

nucleation is the sum of contributions by primary nucleation and second nucleation.

Primary nucleation : occurs in the absence of crystals, secondary nucleation: attributed to the influence of existing crystals

Primary nucleation can be either homogeneous (no foreign particles are present) or heterogeneous (foreign particles present during heterogeneous nucleation)

Rate of primary nucleation has been modeled by the following power law expression:

Crystallization PrinciplesCrystallization Principles

B: number of nuclei formed per unit volume per unit time; N: number of nuclei per unit volume; kn : rate constant; c: instantaneous solute concentration; c*: solute concentration at saturation. (c-c*) term : supersaturation, the exponent of n can range up to 10 but typically is in the range of 3 to 4.

Crystallization Crystallization PrinciplesPrinciples

Two types of secondary nucleation : shear nucleation (occurs as a result of fluid shear on growing crystal faces), contact nucleation ( happens because of crystals colliding with each other and with the impeller and other vessel internal surfaces.

Rate of secondary nucleation in crystallization is the following:

(2)

k1 : rate constant; MT : suspension density, b : can range up to 5 but has a most probable value of 2; j: ranges up to 1.5

with 1 being the most probable value

Crystallization PrinciplesCrystallization Principles

Figure 1: Typical phase diagram. The components in solution consist of the product (ordinate) and the

precipitating reagent (abscissa). The lines with arrows out line one possible way of performing the

crystallization.

- The supersaturation must be above the a certain value before nucleation will begin

- Metastable region : the supersaturation is low that nucleation will not start

- Once the supersaturation has been raised enough to be in the labile region, nucleation can begin.

- At this point, crystals begin to grow, and the supersaturation decreases

- If the supersaturation becomes too high, the nucleation rate will be too great, and amorphous precipitate will result.

Crystallization PrinciplesCrystallization PrinciplesNucleation

SupersaturationSupersaturation

Precipitatant concentration (salt, PEG etc.)

Prot

ein

conc

entra

tion

Under-saturation(protein remains soluble; crystals dissolve)

Nucleation zoneNucleation zone

Precipitation zonePrecipitation zone

Solubility curve

Metastable zoneMetastable zoneCrystals grow, butCrystals grow, but

Nuclei form only Nuclei form only infinitely slowlyinfinitely slowly

[Precipitatant]

Prot

ein

conc

entra

tion

NucleationNucleation

PrecipitationPrecipitationMetastable Metastable

Start w/ soluble protein (undersaturated or metastable)

NucleatNucleates herees here

Incre

ase [

prot

ein],

[pre

cipita

nt]

Crystal growsCrystal growsSequesters Sequesters

proteinprotein[protein] drops[protein] dropsCrystal stops Crystal stops

growing @ solubility growing @ solubility curve curve

Expt incr. [protein], [precipitant]

Xtl grows again, until hits curve

Repeats as follows solubility curve

Crystal GrowthPost nucleation process in which molecules in solution are

added to the surface of existing crystalsThe rate of mass deposition R during crystal growth is:

Overall linear growth rate can also be expressed as:

L : characteristics single dimension of the crystal, such as length

Crystallization PrinciplesCrystallization Principles

(3)

(4)

W: mass of crystals per volume of solvent; A : the surface area of crystals per volume of solvent; kG : overall mass transfer coefficient (depends on temperature, crystal size, hydrodynamic conditions, the presence of impurities); g : usually 0 and 2.5

Crystallization Crystallization PrinciplesPrinciples

Crystal growth is a process that consists of two steps in series – diffusion and surface integration

When the exponents are unity, combining

Equation 3, 5, 6 gives

(5)

ci : concentration at the interface between the liquid and solid phase; kd and kr : mass transfer coefficients

(6)

(7)Thus, if surface integration is very fast compared with bulk diffusion, then kr >> kd, and kG , kd.

Yields and Heat and Material Yields and Heat and Material Balances in CrystallizationBalances in Crystallization

Yields and material balance in crystallizationThe solution (mother liquor) and the solid crystals are in

contact for enough time to reach equilibrium. Hence, the mother liquor is saturated at the final temperature at the final temperature of the process, and the final process, and the final concentration of the solute in the solution can be obtained from the solubility curve.

The yield can be calculated knowing the initial concentration of solute, the final temperature, and the solubility at this temperature.

In making the material balances, the calculations are straightforward when the solute crystals are anhydrous. Simple water and solute material balances are made. When the crystallizations are hydrated, some of the water in solution is removed with the crystals as a hydrate.

Properties of protein crystals

Soft, easy to crushContain large solvent channels

Relatively large organic and inorganic molecules can diffuse inside

Anisotropic physical propertiesBirefrigence due to anisotropic refraction

indicesAbility to diffract X-ray due to regular

spaced lattices

New Techniques: RecrystallizationRecrystallization: method of purifying an organic

solid

Gravity Filtration: method of removing insoluble impurities from recrystallization solution

Suction Filtration: method of isolating pure solid from liquid (filtrate) using vacuum

In LabRecrystallization

Determine best solvent for recrystallizationWeigh crude solidRecrystallizeWeigh purified product

After LabLet recrystallized solid air dry on shelf.Weigh dry crystals and take a melting point (next

week).

Calculations:% recovery: [mass crude/mass recrystallized] x 100

Conclusions: Discuss effectiveness of recrystallization process in terms of purity and percent recovery. Discuss role/attributes of solvent, sources of error, etc.

Pure solid: Tight crystal lattice Impurities disrupt the

crystal lattice

Steps in Recrystallization1. Dissolve crude solid in hot solvent (saturated

solution)

2. Let solution cool to room temperature, so that crystal lattice reforms.

3. Cool solution in an ice bath.

4. Suction filter the pure solid, leaving impurities in solution.

5. Let solid air dry to remove traces of solvent.

4. Suction filter solid away from impurities.

5. Let crystals air dry.

An Ideal Recrystallization Solvent Shouldshould dissolve all of the compound when the solvent is

hot (boiling).should dissolve none of the compound when the solvent

is at room temperature.should have different solubilities for the compound and

the impurities.should have a lower boiling point than the melting point

of the compound.should have a fairly low boiling pointshould be cheap, non-toxic, non-reactive, and non-

smelly

Crystallization

Nucleation

Crystallization and Nucleation