Crystallization of biological macromolecules · Crystallization X-ray diffraction analysis Structure determination Introduction X-ray Crystallography is necessary for a obtaining

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