Transcript Protein Crystallization Theory & Practice

Protein Crystallization
Theory & Practice
Original document:
developed by Tristan Fiedler for
2003 ACA Summer School in Macromolecular
Crystallography
Augmented 6 July 2004 by Andy Howard
Rebuilt for Biology 555, spring 2008
krystallos?
krustallos?
krustallos
Journalist’s Criteria
Who
What
When
Where
Why
How
Whither
Whence
(Wherefore)
Who makes
macromolecular crystals?
Macromolecular crystals are
almost always produced
artificially, i.e., by human
action
So “who” is “scientists”
QuickTi me™ and a
T IFF (Uncompressed) decompressor
are needed to see thi s pi cture.
What are macromolecular
crystals?
Crystals are translationally ordered arrays of
molecules
Macromolecular crystals are held together by
relatively weak ionic intermolecular forces
Solvent content generally above 40%
When have we made them?
Story goes back into the mid-19th century
Systematic search for crystallization
conditions dates from the 1950’s
Screening kits: concept 1980’s,
commercialization in 1990’s
Robotics late 1990’s
Nanoscale techniques early 21st century
Crystallization
Very old (reproduction, genetic
duplication)
Empirical (trial & error -- ‘Screens’)
‘Art’ vs ‘Science’
Short History
1946 Nobel (Chemistry)
1853 Hemoglobin
1926 Urease
Lehman, CG. Lehrbuch der physiologische Chemie. Leipzig
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 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…
Growing [Protein] Crystals
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]
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
r(xtal) = r(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 are
Polar
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
Protein Preparation
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 Strategy
Optimize Protein Expression
Preparation of soluble cell-free extract
AMS/PEG fractionation
Affinity Chromatography
Ion Exchange Chromatography
Size Exclusion Chromatography
Homogeneity Analysis (SDS-PAGE, MS,
DLS)
Protein vs Salt
Property
Salt
Protein
Size
cm’s
< 1mm
Integrity
Electrostatic
Charged Ions
Solvent content
Lower
Fragile ? (needle test)
Less
larger often twinned
Hydrogen bonds
Hydrated Molecules
Higher
allows ligand access & activity
More
Keep Protein Crystals Hydrated
in “Mother Liquor”
Protein Preparation
Protein Storage
Oxidation, Deamination, Denaturation,
Proteolysis, Aggregation
General Rule : Store [x] & purified
> 1 mg/ml
Reducing agents, in vivo pH
Keep on ice / quick freeze in aliquots
Filter sterilize, Antimicrobial agents
Solubility Curve
Below S - no ppt
Zone 1 - Metastable
rare nucleation
sustains growth (seed)
Zone 2- Nucleation
crystals 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 a
TIFF (Uncompressed) decompressor
are needed to see this picture.
What do we do with that?
It can be measured through static lightscattering and SANS measurements
Good correlation with nucleation
conditions, at least with favorable
proteins
Crystallization Methods
Batch
Dialysis
Vapor Diffusion
Dialysis
Double Dialysis
Vapor Diffusion
Vapor Diffusion Variants
Factors affecting Crystallization
Physical:
Temp, pressure, surface, viscosity, vibration
Chemical:
pH, precipitant, ionic strength, metals
Biochemical:
purity, ligands, post-TL, proteolysis
Engineering:
solubility, fusion proteins, heavy atom sites
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
Precipitates
http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html
Good - nonamorphous, birefringent,
redissolves
Bad - skin, does not redissolve,
characteristic brownish tinge
http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html
Whence came we?
It used to be really hard:
Inadequate quantity
Inadequate purity
Unsystematic approaches
Macro quantities required
Motivation to improve crystallization
approaches came as the field matured
Whither?
High-throughput
Better protein purity
Higher quantities when required
Approaches that don’t require large quantities
have appeared
More systematic approaches
Automation at every stage, including
visualization
Automated crystallization
Sample loading, distribution
visualization, decision-making
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (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
References
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.)