Growing protein crystals
This presentation is the property of its rightful owner.
Sponsored Links
1 / 29

Growing Protein Crystals PowerPoint PPT Presentation


  • 114 Views
  • Uploaded on
  • Presentation posted in: General

Growing Protein Crystals. Using Calcium-Integrin Binding Protein as a Model Presented by Chad Blamey. FBP www.scripps.edu/~arvai/ xtals/xtals.html. Goals. What are good crystals Why getting good crystals is important Understand how crystals grow

Download Presentation

Growing Protein Crystals

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Growing protein crystals

Growing Protein Crystals

Using Calcium-Integrin Binding Protein as a ModelPresented by

Chad Blamey

FBP

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


Goals

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

2l

2l

4l

Lysozyme Demonstration

Buffer

30% w/v Polyethelene glycol 5000

1 M NaCl

50 mM NaAcetate pH 4.5

Lysozyme Protein

100mg/ml

50 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

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

Precipitation

Crystalline


Crystallization needs

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

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

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

Good vs Poor Data

Ca+007

M035

4.5 Å

Poor, smeary spots

Notice ‘twined’ spots

4.1 Å

Good! Round spots

Higher order visible (circle)


Spot prediction

Spot Prediction

Crystal M035

Do spots match mathematical predictions?


How do proteins crystallize

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

+

precipitant

+

-

-

-

-

polyethelyene glycol

salts

sugars

organic solvents


Growing crystals hanging drop method

[X]

[Y]

Growing Crystals: Hanging Drop Method

crystals

  • Widely used

  • Vapor diffusion

  • Drop equalizes with reservoir

  • Volume of drop slowly decreases

  • Protein concentration slowly increases

  • CMCC Figure 3.2

drop

reservoir

Sitting drop


Phases of proteins in solution

Crystals

Figure 3.3

Precipitation

Metastabile

Supersaturation

Growth & Nucleation

Solubility

Barrier of Nucleation

Undersaturated

Growth only

[Protein]

Soluble protein

Phases of Proteins In Solution

Not to be confused with phases of light

CMCC figure 3.3


Nucleation growth

Phase diagram

Figure 3.3

Metastabile

Supersaturation

Solubility

Undersaturated

[Protein]

Nucleation & Growth

Basic concept:

  • Concentrate solution enough so nucleation occurs in only a few cases

  • Initial growth pulls some protein out of solution

  • Reducing [protein] back into metastable range

  • Grow only a few large crystals


Optimize crystal growth

[X]

[Y]

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


The tricky part

The Tricky Part

  • Conditions for crystallization are dependent on each-other

  • Crystal quality will change as you vary growth conditions

  • Figure 3.4

[B]

[A]

For solution made up of three parts A, B and C.

Changing [C] will effect the quality of the crystal in

terms of [A].


Growing crystals other techniques

Spin

6 hours

12 hours

0 hours

Growing Crystals:Other Techniques

Ulatacentrifugation

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

  • Slowly increases the relative protein concentration

Dialysis

  • Uses liquid-liquid diffusion

  • Diffusion is slow

  • Rate controlled by membrane


Crystal screens

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

Factors Effecting Crystal Growth

*Most important


Characterization of protein

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

CIB purified w/ reducing agents

CIB purified w/o reducing agents

IEF

Western-

Blot

IEF

SDS-PAGE

Marker

No DTT

DTT

CIB

pH 5.6

22

CIB Protein Characterization

Further characterization of a protein can

improve purity and therefore crystal quality


Ligands and co crystallization

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

Additives

  • Often designed to reduce strength of protein-protein interaction

  • Detergents important category

  • Reducing agents

  • Organic solvents


Small crystals

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

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

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

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

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?


Growing protein crystals

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

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?


  • Login