recent developments in protein crystallisation andrzej marek brzozowski york structural biology...
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Recent developments in protein crystallisation
Andrzej Marek Brzozowski
York StructuralBiology Laboratory
Dodsonium eleanoraeDodsonium adalbertus (common sp. name: guy)
• Highly infectious • No real immediate remedy •Very limited chances of disinfection as virulance factors are easily
soluble in ethanol derivatives (esp. virulence factors of D. adalbertus)• Frequent reoccurrence of symptoms
They attack and paralyse central nervous system:
-making the sufferer unable to have a clear judgement of everyday situations
-wiping out the remainings of the free will
-cataract-like effect resulting in a total cloudiness of the objectivity
-the day-by-day growing amount of confusion and chaos leads to a total acceptance of their vision of the world as the only remedy to survive
However, the main side-effect is that nobody wants to be cured!
..as the prolonged infection creates in the sufferer feelings of his/her uniqness
unlimited capabilities,readiness to solve any scientific and life problem,
full harmony with the beauty of life, human kind, earth, Universe
and beyond
Main components of protein crystallisation:
• Theoretical foundations of nucleation and crystallisation
• protein as a variable
• crystallisation hardware
• crystallisation supports (plates etc.• enhancement of nucleation• robots• crystal handling• image recognition systems
• chemistry (screens, precipitants)• matrices• chemicals
Tc
phase separation
criticaltemperature
L1 branch L2 branch
benzene concentration0 % 100 %
T
Phase diagram of benzene / water
Phase separation
No phase separation
Courtesy of Jan Drenth
T ( C)O
lysozyme concentration (mg/ml)
Tc
L branch1
L branch2
10
100 200 300 4000 500crystalline state
Phase diagram of lysozyme
solubility curve
Courtesy of Jan Drenth
In short, second viral coefficient: B22 reflects (through W22 (potential of mean force)) the total thermodynamic environment for protein molecules diluted in a given solvent
George A. & Wilson W.W. Acta Cryst (1994), D50, 361-365
Liquid-Liquid phase separation: coexistence of dilute & dense liquid
Liquid-Crystal phase separation: coexistence of protein solution & crystals
Ten Wolde & Frenkel, Science (1997), 277, 1975-1978
If the range of attraction is reduced (even below 25% of the colloid diameter)
Crystallisation near the metastable fluid-fluid critical point is strongly influenced by the large density fluctuations that occur in the vicinity of such a critical point.
The critical density fluctuations around Tc lead to a streaking change in the Free-energy landscape.
The crystal nucleation barrier may be lowered by:- adjustment of the solvent conditions (for instance by the
addition of the nonionic polymer) therebychanging the range of interaction
- crystallisation near Tc!!! (reduction of G by ~30kBT increases the nucleation rate by ~1013)
One can selectively speed up the rate of crystal nucleation, without increasing the rate of crystal growth, or the rate at which amorphous aggregates form.
This phenomenon occurs both in the bulk and in (quasi) two-dimensional systems (such a membranes). So, nature already makes extensive use of critical density fluctuations to facilitate the formation of ordered structures.
Nucleation mechanisms
Haas C. & Drenth J. J. Cryst. Growth (1999), 196, 388-394
The route to the critical nucleus leads through a formation of a liquid-like droplet, in which, beyond a certain critical size, crystalline nucleus is formed.
One of the possible explanation of this is that the wetting of the crystal nucleus by a liquid-like layer results in a value of a interfacial energy (g), which, in consequence lowers the DG* barrier (Haas & Drenth, 1995, J Cryst.Growth, 154, 126)
Liquid-Liquid phase separation: coexistence of dilute & dense liquid
Liquid-Crystal phase separation: coexistence of protein solution & crystals
Ten Wolde & Frenkel, Science (1997), 277, 1975-1978
If the range of attraction is reduced (even below 25% of the colloid diameter)
PROTEIN AS VARIABLE
• new expression system (cell free systems)
• automation of expression
• protein tagging/fusion
• protein surface/crystal contacts engineering
YkoF
RGSL domain of PDZRhoGEF
Lcrv antigen
Rational protein surface/crystal contacts engineering
- Rational protein surface engineering is an effective crystallisation strategyleading to the favourable crystal contact formation and minimisation of protein disorder
- clusters: K→A , E→A, E→Q, other mutations?
Derewenda Z.S. (2004) Structure,12, 529-535
Cherezov V. & Cafferey M. (2003) J Appl. Cryst., 36, 1372-1377
CRYSTALLISATION HARDWARE
• crystallisation supports (plates etc.)
Microplates for the cubic lipidic phases
Hansen C.L. et al. (2002) PNAS, 99, 16532-16536
• 48 precipitant wells – 3 L aliquotes• 3 L protein sample load
Crystallisation chips I
CRYSTALLISATION HARDWARE CONT.
Crystallisation chips II
Bo Zheng at al. (2004) Angew. Chemie Eng. Int. Ed., 43,2508-2511
ENHANCEMENT OF PROTEIN NUCLEATION
• transistor devices with carbon nanotubes for charge transfer studiesBradley K. et al. (2004) Nanoletters, 4, 253-256
• designer self-assembled nanotubes Hill J.P. at al (2004) Science, 304,1481-1483
Laser irradiated growth of protein crystals
Adachi H. et al. (2003) Jpn. J. Appl. Phys., 42, L798-L800
Pre-stirring promotes nucleation of protein crystals
Adachi H. et al. (2004), Jpn. J. Appl. Phys., 43, L243-L246
Kondepudi et al. (1990) Science, 250,975-976 (Chiral symmetry breaking in sodiumChlorate crystallization)
CRYSTAL HANDLING
Kiefersauer R. et al. (2000) J Appl. Cryst. 33, 1233-1230
Yao M. et al. (2004),Acta Cryst., D60, 39-45
Sjögren T. et al. (2002)J Appl. Cryst., 35, 113-116
Automated analysis of vapour diffusion drops with an X-ray beam
Jaquamat L. et al (2004) Structure, 12, 1219-1225
CRYSTALLISATION ROBOTS
• Commercial: Cartesian, Tecan, Douglas,Mosquito,Hydra, Gilson (Cyberlab), Decode Biostructures (ROBOHTC))
• Custom made: Syrrx (Agincourt system), Affinium Pharm. (Gem system (Cartesian based)), Hauptman-Woodward Med.Res.I. (1536 system). Protein Structure Factory (Berlin), EMBL Grenoble
IMAGE RECOGNITION SYSTEMS
• detection of crystalline elements in crystallisation droplets using rotating polarizer microscope technique
Echalier A. et al. (2004) Acta Cryst., D60, 696-702
• seven broad pH range (4-10) three components buffers systems assuring wide pH range, chemical variability and small number stock solutions (14)
Newman J. (2004) Acta Cryst., D60, 610-612
CRYSTALLISATION CHEMISTRY
• warnings!… unexpected effects of some folding stabilisers (NDSBs)
The effect of TMAO and NDSB 201 on a test protein
TMAO, NDSB 201 [mM]
CONCLUSIONS (if any):
•Need for a tighter interface between theoretical studies and experimental science
•Innovations in crystal handling could be a new breakthrough in improvement of its quality and diffraction properties
•More crystallisation oriented and more universal robots are still required
•The chemistry of precipitants, gels, stabilising solutions etc. still has to developed – striking lack of progress in last ~15 years
“Please step forward to the rear”
(Approximate translation of a request once heard in tram-car in Warsaw.From ‘How to be a Conservative-Liberal-Socialist’ by L. Kolakowski)
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