Growing Protein CrystalsUsing Calcium-Integrin Binding

Protein as a ModelPresented by

Chad Blamey

FBPwww.scripps.edu/~arvai/ xtals/xtals.html

Goals

• What are good crystals– Why getting good crystals is important

• Understand how crystals grow• Discus techniques for crystallizing proteins

– Application type of discussion

• Strategies for optimizing crystal growth– Understanding your favorite protein

• CIB as a model– My favorite protein!!!

• Lysozyme demonstration

Lysozyme Demonstration

2l 2l

4l

Buffer30% w/v Polyethelene glycol 50001 M NaCl50 mM NaAcetate pH 4.5

Lysozyme Protein100mg/ml50 mM Na Acetate pH 4.5

glass slide

Should make large crystals in less than 15 minutes.We will watch it for the hour of the lecture.

Everyone Should Know

• Protein crystals are precipitated protein in solutions

• You can think of them highly concentrated aqueous solutions (usually about 500 mg/ml)

• Amorphous precipitation is random• Crystals are ordered

– This is the property we are interested in– Figure 3.1 CMCC– Gray areas!

Crystals PrecipitationCrystalline

Crystallization: Needs

• Obtaining quality crystals is by far the limiting step to solving a structure

• Crystals need to be of sufficient size and quality to diffract x-rays

• Size: Normally should be 100 m in smallest dimension• Quality: Reflections collected from diffraction data are the

primary source of data to build an electron density map, therefore quality of protein model depends greatly on crystal quality– Growing good crystals is key to a good structure

Crystallization

• With enzymes is is often important to maintain enzymatic activity in crystal– Some enzymes can function in crystal

• Best way to test crystal quality is by mounting a crystal and attempting to diffract x-rays– Visual inspection helpful too– May not be meaningful

Low vs High Data

• Difference between 9.0 Å and 4.5 Å

• The higher the resolution the better!

• CIB crystal spots

9 Å

4.5 Å

Good vs Poor Data

Ca+007 M035

4.5 ÅPoor, smeary spots

Notice ‘twined’ spots

4.1 ÅGood! Round spots

Higher order visible (circle)

Spot PredictionCrystal M035

Do spots match mathematical predictions?

How Do Proteins Crystallize?

• For crystallization to occur it has to be thermodynamically favorable

• Precipitants remove available water forcing proteins to associate with each other

• Hopefully in a organized fashion

Water

Protein

polyethelyene glycolsaltssugarsorganic solvents

precipitantprecipitant

- --

-

++

++

Growing Crystals: Hanging Drop Method

• Widely used

• Vapor diffusion

• Drop equalizes with reservoir

• Volume of drop slowly decreases

• Protein concentration slowly increases

• CMCC Figure 3.2 A1 A2 A3 A4 A5 A6B1 B2 B3 B4 B5 B6C1 C2 C3 C4 C5 C6D1 D2 D3 D4 D5 D6

[X]

[Y]Sitting drop

reservoir

drop

crystals

Phases of Proteins In SolutionNot to be confused with phases of light

[Protein]

So

lub

ility

SupersaturationMetastabile

Undersaturated

Precipitation

Crystals

Growth & Nucleation

Growth only

Barrier of Nucleation

Soluble protein

Figure 3.3

CMCC figure 3.3

[Protein]

So

lub

ility

SupersaturationMetastabile

Undersaturated

Figure 3.3 Phase diagram 1. Concentrate solution enough so nucleation occurs in only a few cases

2. Initial growth pulls some protein out of solution

3. Reducing [protein] back into metastable range

4. Grow only a few large crystals

Basic concept:

Nucleation & Growth

Optimize Crystal Growth

• The number of factors can be overwhelming

• Focus on those factors which most effect growth

• Set up arrays to vary two different conditions at once

• Cross your fingers

A1 A2 A3 A4 A5 A6B1 B2 B3 B4 B5 B6C1 C2 C3 C4 C5 C6D1 D2 D3 D4 D5 D6

[X]

[Y]

Crystal Quality

The Tricky Part

• Conditions for crystallization are dependent on each-other

• Crystal quality will change as you vary growth conditions

• Figure 3.4[A]

[B]

For solution made up of three parts A, B and C.Changing [C] will effect the quality of the crystal interms of [A].

Growing Crystals:Other Techniques

• Spin at extremely high speeds, hundreds of thousands of g’s

• Slowly increases the relative protein concentration

Spin

0 hours 6 hours 12 hours

Ulatacentrifugation

Dialysis•Uses liquid-liquid diffusion•Diffusion is slow•Rate controlled by membrane

Crystal Screens

• Hampton Research screen tests a wide assortment of conditions of salts, buffers, pH’s and additives– Best conditions from

literature

• Often first hits with screens are small poor quality crystals

• Do not use the absence of crystals as a gauge of conditions rather use solubility

Factors Effecting Crystal Growth

• Ionic Strength*

• Specific Ions (Ca2+)

• Protein Concentration*

• Detergents

• Inorganic Precipitant

• pH*

• Temperature*

• Time

• Monodispersion*

• Vibrations

• Pressure

• Gravity

• Relative Proportion of Conditions

• Purity Of Protein*

• Access to water*

• Ligands

• Binding partners

*Most important

Characterization of Protein

• Of course the more you know about your protein the easier it is to manipulate

• Cystine is often the most critical a.a.– CIB has three and no disulfide bonds, but cause multimers

• Key ligands and metals, like Ca (for CIB)• Stability in certain solutions

– Hydrodynamic radius (NMR)– Stability (CD is great)– Dynamic light scattering– Mass spec

CIB Protein Characterization

IEFWestern-

Blot

IEF CIB purified w/o reducing agents

SDS-PAGENo DTT DTT

Mar

ker

22CIB

pH 5.6

CIB purified w/ reducing agents

Further characterization of a protein can improve purity and therefore crystal quality

Ligands and Co-crystallization

• Try to obtain crystals with different ligands and/or co-crystallize with another protein– Metals, peptides & binding proteins– Proteins with a known structure can simplify the process

• Enzymes and Substrate complexes– Non-competitive inhibitors– Substrate analogs

• Often changes protein conformation– Two structures!– This gives information about how the protein function

Additives

• Often designed to reduce strength of protein-protein interaction

• Detergents important category

• Reducing agents

• Organic solvents

Small Crystals

• Often small crystals can be made larger by microseeding new drops with previously grown crystals or adding more protein solution

• Multinucleation can be avoided by reducing the temperature or adding glycerol

• Crystals only need to be large enough to diffract x-rays well

Radical Approaches

• Remove either N- or C-terminus by weak proteolysis or by molecular cloning– Often termini can be disordered which interferes with lattice

formation

• Crystallize with a fusion protein– Fusion proteins are well documented with a solved structure

that easily from a lattice, example: GST– “Pull” the fusion protein into an ordered crystal– Can use the protein for molecular replacement to solve phase– Many recombinant proteins are purified using fusions

anyways, i.e. not hard to try

Radical Approaches, Cont

• Mutants:– Specific residues problem residues can be mutated

using recombinant DNA technology

– Domains can be crystallized separately

• Issues:– Different conformations from the native state likely

– Domains can only be part of the story

– Changing the means starting over in terms of crystallization solution

Discussion

• Given a hypothetical protein that doesn’t give you any positive hits in your first screen what could you do to obtain quality crystals?– Meaning: almost no precipitation in each drop!

• Likewise what if you get lots of precipitation?• Say on the other hand at room temperature you

have a condition with lots of tiny crystals, what can be done to reduce the amount of nucleation?

Lysozyme

• Vapor diffusion takes about 12 hours to complete. Where on phase diagram did the [lysozyme] start given almost no vapor diffusion occurred?

• Lysozyme in other solutions crystallizes much more slowly (period of days). Which solutions would yield higher quality crystals? Why?

• What are some of the properties of lysozyme that allow it to crystallize so quickly?

CIB

• Similar to a ubiquitous protein Calmodulin

• Binds calcium• Regulates other

proteins (13 so far)• Found in most tissues

types: brain, muscle etc.

• No enzymatic activity

CIB Discussion

• What are some possible techniques that could be used to obtain CIB crystals?

• What about the cystines of CIB?• Why is it important that the radius of CIB is

smaller when it is bound to calcium?• CIB contains a surface exposed hydrophobic

patch, how could this information change your crystallization conditions?