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Transcript of Original document: developed by Tristan Fiedler for 2003 ACA Summer School in Macromolecular...
Original document:developed by Tristan Fiedler for
2003 ACA Summer School in Macromolecular Crystallography
Augmented 6 July 2004 by Andy HowardRebuilt for Biology 555, spring 2008
Protein CrystallizationTheory & Practice
krystallos?krustallos?
Journalist’s Criteria
WhoWhatWhenWhereWhyHow
Whither
Whence
(Wherefore)
Who makes macromolecular crystals?
Macromolecular crystals are almost always produced artificially, i.e., by human action
So “who” is “scientists”
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
What are macromolecular crystals?
Crystals are translationally ordered arrays of moleculesMacromolecular crystals are held together by relatively weak ionic intermolecular forcesSolvent content generally above 40%
When have we made them?
Story goes back into the mid-19th centurySystematic search for crystallization conditions dates from the 1950’sScreening kits: concept 1980’s, commercialization in 1990’sRobotics late 1990’sNanoscale techniques early 21st century
Very old (reproduction, genetic duplication)
Empirical (trial & error -- ‘Screens’)
‘Art’ vs ‘Science’
Crystallization
1853 Hemoglobin Lehman, CG. Lehrbuch der physiologische
Chemie. Leipzig
1926 Urease Sumner, JB. J Biol Chem. 69: 435
1930 Pepsin & other proteolytic enzymes Northrop, JH, Kunitz, M, Herriot RM. Crystalline Enzymes. Columbia
University Press, NY (review)
1934 Pepsin Diffraction Bernal JD & Crowfoot, D. Nature,
133:794
1935 Tobacco Mosaic Virus Stanley, WM. Science. 81:
644
Short History1946 Nobel (Chemistry)
Short history (concluded)
1946: Sumner Nobel prize
1958: Myoglobin structure
1959: Hemoglobin structure
1962: Perutz/Kendrew Nobel
1979: Carter&Carter paper
1985: first microgravity experiments
1990’s: commercial screening kits
Late 1990’s: viable commercial crystallization robots
Where do we grow them?
Under mild laboratory conditions
Contrast to inorganic small molecules, which are often grown from a melt
Even small organic crystals often exploit temperature dependence
Proteins usually avoid these techniques…
Inorganic - cooling a hot saturated substance
Polar organic - same or ppt from aqueous using organic solvents
Proteins - Yeoww!! (denature)
1.Dissolved in buffer + ppt [low]
2.controlled evaporation [higher]
Growing [Protein] Crystals
Why do we grow them?
Because we want to know the macromolecule’s structure!
Fundamental postulate:The structure of a protein in a crystal differs only slightly, and then only on the surface, from that its soluble or membrane-associated (biologically active) form
Do we believe this?
Short answer: yes
Skepticism was rampant through the 1970’s and has only gradually diminished
Various experimental demonstrations
Evidence that(xtal) = (solution)
Enzymatic activity of crystals (1970’s)
Similarity of multiple crystal forms
Comparisons to NMR structures
Consistency with other biophysical techniques
When is the postulate wrong?
Some external loops held in wrong positions (Interleukin-8)Much more common: crystal structure shows us one conformer; other conformers, and the transitions among them, are relevant
How to make 3-D crystals
In general it involves creation of three-dimensional order
In practice with macromolecules that means creating conditions in which intermolecular forces can be exerted in the same way on each molecule
These intermolecular forces arePolar
Often water-mediated
Weak
How do we grow macromolecular crystals?
Short answer: we gradually decrease the solubility of the protein in a way that produces ordered (crystalline) precipitation rather than disordered (amorphous) precipitation
Recognize the stages in crystallization
Stages of crystallization
Nucleation
Governed by short-range intermolecular interactions
We want a few stable nuclei, not a lot!
Growth
Adding one molecule at a time to the nucleus
Incorrect additions lead to instability and…
Cessation of growth
Know your protein
Cysteines
Substrates / Ligands
Proteolytic sensitivity
Metal binding
pH & Temp for stability / activity
Post Translational Modification
Protein Preparation
Purify from natural sources
Create an expression construct
Add Tags to aid purification
6-His, Biotin-Strept., Calmodulin Bind. Peptide, GST, Maltose Bind. Protein
Expression systems
E.coli - no post translational modifs
Yeast - euk, may be better for secreted pro’s
Baculovirus-Insect & Mammalian cells
Protein Preparation
Purification StrategyOptimize Protein ExpressionPreparation of soluble cell-free extractAMS/PEG fractionationAffinity Chromatography
Ion Exchange ChromatographySize Exclusion ChromatographyHomogeneity Analysis (SDS-PAGE, MS, DLS)
Protein Preparation
Protein vs SaltProperty Salt Protein
Size cm’s < 1mmlarger often twinned
Integrity ElectrostaticCharged Ions
Hydrogen bondsHydrated Molecules
Solvent content LowerHigher
allows ligand access & activity
Fragile ? (needle test)
Less More
Keep Protein Crystals Hydratedin “Mother Liquor”
Protein StorageOxidation, Deamination, Denaturation, Proteolysis, AggregationGeneral Rule : Store [x] & purified> 1 mg/mlReducing agents, in vivo pHKeep on ice / quick freeze in aliquotsFilter sterilize, Antimicrobial agents
Protein Preparation
Solubility CurveBelow S - no ppt
Zone 1 - Metastablerare nucleationsustains growth (seed)
Zone 2- Nucleationcrystals grow
Zone 3 - Precipitation
Does it always work this way?
No. Some proteins are more soluble in high salt than low.
Same general principles apply as long as we understand the dynamics
What does aggregation do?
In a sense, a crystal is an aggregate…
The formation of oligomeric, randomly oriented aggregates is not conducive to crystallization
We’ll see a useful tool next week for detecting aggregation
Second virial coefficient
Characterizes two-body interactions between protein molecules in dilute solution
QuickTime™ and aTIFF (Uncompressed) decompressor
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What do we do with that?
It can be measured through static light-scattering and SANS measurements
Good correlation with nucleation conditions, at least with favorable proteins
Batch
Dialysis
Vapor Diffusion
Crystallization Methods
Dialysis
Double Dialysis
Vapor Diffusion
Vapor Diffusion Variants
Physical:Temp, pressure, surface, viscosity, vibrationChemical:pH, precipitant, ionic strength, metalsBiochemical:purity, ligands, post-TL, proteolysisEngineering:solubility, fusion proteins, heavy atom sites
Factors affecting Crystallization
Don’t be deceived!http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial1.html
Beautiful - No diffraction
Ugly - 1.6 Angstroms !
Moral : Its the diffraction that counts
Good - nonamorphous, birefringent, redissolves
Bad - skin, does not redissolve, characteristic brownish tinge
Precipitateshttp://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html
http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html
Whence came we?
It used to be really hard:Inadequate quantityInadequate purityUnsystematic approachesMacro quantities required
Motivation to improve crystallization approaches came as the field matured
Whither?
High-throughputBetter protein purity
Higher quantities when required
Approaches that don’t require large quantities have appeared
More systematic approachesAutomation at every stage, including visualization
Automated crystallization
Sample loading, distribution
visualization, decision-making
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Can we get away with not knowing our protein?
Often, yes: cf. Structural genomics projects
Ugly cases (e.g. transmembrane): we still argue that the more you know, the more likely you are to get good crystals
www.hamptonresearch.com
www.emeraldbiostructures.com
Protein Crystallization (ed. T. Bergfors) http://xray.bmc.uu.se/~terese/
Crystallization of Nucleic Acids and Proteins (ed. A. Ducruix & R. Giege)
http://www.hwi.buffalo.edu/High_Through/High_Through.html
International Tables for Crystallography. Vol. F
Part 3 : Techniques of Molecular Biology (S. Hughes & A. Stock)
Part 4 : Crystallization (R. Giege et al.)
References