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Student: Tomáš Kučera
Project leader: Ph.D. student Ekaterina Sviridova
Summer academical course 2010Institute of Physical Biology
Nové Hrady, Czech Republic
THIS PROJECT HAS RECEIVED FINANCIAL SUPPORT FROM THE EUROPEAN SOCIAL FUND AND FROM GOVERNMENT OF THE CZECH REPUBLIC
Crystallization of biological macromolecules
Aims of the project
To learn strategy, techniques, materials and parameters for setting up crystallization trials on the model protein such as lysozyme and thermolysin
To determine optimal crystallization conditions for Fe-regulated protein D (FrpD)
To learn COOT program for electron density map modifications
X-ray diffraction analysishttp://www.chem.ufl.edu/
X-ray Crystallography stages:
Cloning, expression and purification
Crystallization
X-ray diffraction analysis
Structure determination
IntroductionX-ray Crystallography is necessary for a obtaining
3D structure of biological macromolecules; it’s a key in understanding of enzymatic or structural function of proteins.
Crystallization
is a process of formation solid crystals from the solution
is a laboratory technique, which help us to solve structure of molecules
Crystallization methods
Vapour diffusion:
hanging drop
sitting drop
sandwich drop
Batch
Microbatch under oil
Counter diffusion in single capillaries
http://www.pdb.org/
Results
Lysozyme Enzyme consisting of 129
amino acids, which damage bacterial cell walls
Molecular weight: 14,3 kDa
Component of animal tissues, organs and serum in tears or milk
The first enzyme structure to be solved by X-ray diffraction methods
Crystallization of lysozyme
Concentration of lysozyme:
20, 40, 60, 80 and 100 mg·ml−1
Precipitant:
5%, 6%, 7%, 8% and 12% solution of NaCl
Methods:
vapour diffusion (hanging drop, sitting drop), batch, microbatch under oil, counter-diffusion
Ratio:
1:1, 2:1, 1:2
Crystallization of lysozyme
▲Hanging drop60 mg.ml−1
10% NaCl1:1
Sitting drop60 mg.ml−1
8% NaCl1:1▼
▲Microbatch under oil60 mg.ml−1
8% NaCl1:2
Counter diffusion
60 mg.ml−1
10% NaCl▼
Crystal analysisDye test
Crush test
Termolysin
Heat-stable methaloproteinase produced by Bacillus thermoproteolyticus
Molecular weight: 34,6 kDa
Specifically catalyzes the hydrolysis of peptide bonds containing hydrophobic amino acids http://www.pdb.org/
Crystallization of thermolysin
Concentration: 25 mg.ml−1
Precipitant composition:
0,14 M Magnesium acetate tetrahydrate
0,1 M Sodium cacodylate trihydrate pH 6,5
20% (w/V) Polyethylenglykol 8000
Ratio: 1:1
Temperature: 18 °C
Fe-regulated protein D (FrpD protein)
FrpD is a highly conserved lipoprotein of Neisseria meningitidis anchored to the bacterial outer membrane
Molecular weight: 30 kDa
Biological function unknown: binds with FrpC protein, probably helps to anchoring of FrpC protein to the bacterial outer membrane
However, mechanism of FrpD-FrpC interaction is unknown due to the absence of any structural information on these proteins
Crystalyzation of Se-Met FrpD
Concentration of protein: 8 mg·ml−1
Precipitant composition:
20% (w/V) PEG 8000
20% (V/V) PEG 400
0,1 M MgCl2
0,1 M Tris pH 8,5
Ratio: 1:1Size: 1,08 × 0,12 × 0,075 mm
COOT: Electron density map modification
41st
residue Glutamic Acid before modification and after
COOT: Electron density map modification
30th
residue Tyrosine before modification and after
Conclusions
Crystals of lysozyme and thermolysin proteins were obtained using different crystallization techniques.
Optimal conditions for Se-Met derived FrpD protein crystallization were adjusted and established.
Electron density map modifications help us to determine 3D structure of biological macromolecules.
Acknowledgement
Many thanks to:
my supervisor Ekaterina Sviridova
Oksana Degtjarik, Tatyana Prudnikova and Katsiaryna Tratsiak
organisers of this course
THIS PROJECT HAS RECEIVED FINANCIAL SUPPORT FROM THE EUROPEAN SOCIAL FUND AND FROM GOVERNMENT OF THE CZECH REPUBLIC
THANK YOU FORYOUR ATTENTION