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Crystallographic structure analysis of Chitinase enzyme from Corms of Crocus vernus
Dr. Ahmed AkremBahauddin Zakariya University, Multan
29.04.14
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Importance of Chitinase
Chitinases catalyze the hydrolysis of chitin1
Chitinases occur in a wide range of organisms, including plants, animals, viruses,
bacteria, fungi and insects, and play a variety of roles in these organisms.2
Plant chitinases are a structurally diverse group with respect to their physical
properties, enzymatic activities and localization.3
Chitin is an unbranched homopolymer of 1,4-linked N-acetyl-d-glucosamine.4
Chitin is not a component of mammalian cells; it occurs widely elsewhere in nature
and is abundant in human pathogens.
More than 75% of the industrial enzymes are hydrolases.5
1Bishop et al., 2000; 2Brunner et al., 1998; 2Hoell et al., 2005; 3Collinge et al., 1993; 4Butt & Sultan, 2010;
5Leishola et al., 2005
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1. Isolation and purification of the chitinase protein from C. vernus
2. Crystallization of the purified protein
3. X-ray diffraction data collection
3D molecular structure determination
Aims and Objectives
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Protein Crystallization
Nextal.com
Non-recombinant
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Crocus vernus
Genus Crocus belongs to family Iridaceae.
A perennial flowering plant found in Central and Southern Europe,
North Africa, Middle East, Central Asia to China.
Most expansive spice “Saffron” is from Crocus sativus L.
Chitinases & Lectins are ´´ Defense-related plant proteins``.
Plant chitinases are a structurally diverse group.3
So far no crystal structure of this plant is deposited in the Protein Data
Bank.
3Collinge et al., 1993,
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Crude protein porfile
Crocus vernus118kDa
66.2kDa
45.0kDa
35.0kDa
25.0kDa
18.4kDa
14.4kDaCrude Protein profile from corm on
SDS-PAGE
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Purified Chitinase
Final optimized Mono S chromatogram on 12% SDS-PAGE produces lectin
contaminations. Size exclusion chromatography produces 30 kDa protein bands with identical pattern
under reduced and non-reduced conditions. Partial N-terminal sequence blast showed 50% identity with the already reported
chitinases.11
T L F V E Y I G Y P L F S G V K F S D V P I N P E I T K F Q
Mono S peak Size exclusion chromatogram
L1: Reduced, L2: Non-reduced
PVDF blot
11Akrem et al., 2011
Purification techniques/Instruments
Protein characterization
• N-terminal amino acid sequencing
• MALDI/TOF Mass spectrometry
• SDS-PAGE Protein Purification
• Ammonium sulfate precipitation
• Dialysis
• Column Chromatography
• Gel filtration
• Ion exchanger columns (Cation/Anion: Isoelectric pH or pI)
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Crystallization of Chitinase
Dynamic Light Scattering (DLS) measurement of the 30 kDa purified protein showing
monodispersive and monomeric protein solution. PCT™ was performed to optimize the protein concentration. Protein crystallized at concentration of 16 mg ml -1.
Vapor diffusion method Crystal with dimensions of 0.625 × 0.370 × 0.1 mm: Scale bar, 0.5 mm. 0.1M CHES, pH 9.0 and 20% (w/v) PEG 800011
Monodisperse RH = 2.6nm Thin sheet
11Akrem et al., 2011
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Vapor Diffusion Methods
Hanging Drop Sitting Drop
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Protein Crystallization
Phase Diagram
Metastable Soluble aggregate formation but no nucleation Nucleation Critical nuclei formation and crystal growth Precipitation No nucleation. Growth of amorphous precipitate
Crystallization Machinery
Dynamic Light Scattering
(Monodispersity)
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Nanodrop (Protein quantification)
Zinsser Pipetting Robot (Digilab Genomic Solution, Germany) UV-microscope
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Protein/Salt Crystals
VIS-microscope UV-microscope 1mm size approx.
Best is to go for diffraction image
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Diffraction to 3D
Diffraction Image
3D StructureSingle CrystalX-ray Bombarment
Crystallographic Softwares
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X-ray Diffraction Data
Diffractometer Rotating anode Synchrotron: the best ultimate choice A synchrotron is a particular type of cyclic particle accelerator in which the magnetic
field (to turn the particles so they circulate) and the electric field (to accelerate the
particles) are carefully synchronized with the travelling particle beam Deutsches Elecktronen Synchrotron (DESY), Hamburg, Germany Approx. 1000 scientists from more than 30 countries around the world are working (2008) Few countries in the world are enjoying this facility
Diamond, UK ESRF, France DESY, Germany
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Crystal Mounting
Nylon loops to fish out crystals
Goniohead
X-ray gun
Cryonozzle
Microscope
Beamstop
Detectors
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Bioinformatics
Imosflm; Scala
Denzo; Scalepack
CCP4i Suite
Molecular Replacment; Molrep, Phaser, Mrbump
Homer
COOT
Refmac5
Protein Data Bank
Pdbsum
Pdb goodies
Chimera
Pymol
Auto-Rickshaw
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X-ray diffraction Data
Space group C2
Unit-cell parameters (Å,°) a = 172.3, b = 37.1, c = 126.4 Å, β = 127o
VM (Å3/ Da) 2.7
Solvent content (%) 54.2
Resolution range (Å) 25.0 - 2.1 ( 2.2 - 2.1 )
Total Reflections 14,0335 (20369)
Unique reflections 36468 (5230)
Redundancy 3.8 (3.9)
Average I/σ (I) 17.2 (6.8)
R merge* (%) 6.2 (19.4)
Data Completeness (%) 96.6 (96.0)
Table 6: Statistics for the native crystal11
Values in parentheses are for the highest resolution shell.
*R merge =∑hkl ∑i | Ii (hkl) – <I (hkl)> | ⁄ ∑hkl∑i Ii (hkl), where <I (hkl)> is the mean intensity of the observations Ii (hkl) of reflection hkl.
11Akrem et al., 2011
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mtz and pdb files
Out put of first processing is a single mtz file of few MB
Electron density map
Second important file is pdb file based on sequence homology from Protein Data Bank (PDB) like INAR for Narbonin vicia
MR strategy to solve the phase information
Sequence identity of atleast 40%
Clustalw 2, www.pdb.org
Pdbgoodies input page
Phase information and Coordinate information
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GH18 Type Chitinase
Phase problem was solved by Molecular Replacement (MR) using the narbonin structure (PDB code: 1NAR) as search model and the program Molrep.
Chitinases catalyze the hydrolysis of chitin. Chitinases occur in a wide range of organisms including plants, animals, viruses,
bacteria, fungi and insects. In glycosyl hydrolases, they are classified into family 18 and family 19 chitinases.13
Family 18, in their catalytic domain, possesses a common α, β-TIM barrel fold. Matthews’s coefficient calculations indicated two molecules per asymmetric unit.
13Henrissat & Bairoch, 1993
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TIM Barrel
Triose Phosphate Isomerase (TIM) Main feature of TIM barrel is an eight stranded parallel β-barrel making a core surrounded
by α- helices. The cavity of the TIM barrel in CVC is filled with aromatic and polar residues. The catalytic motif of CVC is directed into the cavity of TIM barrel.
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Sequence alignment between CVC, Heavime & Narbonin
16% sequence identity between CVC
and Hevamine (2HVM).14
33% sequence identity between CVC
and Narbonin. Hevamine: a plant endochitinase
isolated from rubber tree (Hevea
brasiliensis). All three proteins are sharing a TIM
barrel structure. Catalytic motif is DXDXE Two consensus sequences have been
highlighted through blue squares.
14Terwisscha van Scheltinga et al., 1996
Sequence Alignment
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Structure Alignment
The superposition of the Cα atoms of CVC with that of other members of the family
gives a rmsd of 3.5 and 3.6 Å for models 2HVM, 1NAR respectively. The catalytic motifs for all structures are on similar position in the TIM barrel. Narbonin, due to lack of an Aspartate in the catalytic motif, cannot show chitinase activity.
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Summary
A 30 kDa chitinase protein was purified from Crocus vernus corm.
Single suitable size crystals were developed from pure enzyme
Already the native Chitinase structures have been deposited at the Protein Data Bank with ID code 3SIM.
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