1 Crystallographic structure analysis of Chitinase enzyme from Corms of Crocus vernus Dr. Ahmed Akrem Bahauddin Zakariya University, Multan 29.04.14
2 Importance of Chitinase Chitinases catalyze the hydrolysis of chitin 1 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 1 Bishop et al., 2000; 2 Brunner et al., 1998; 2 Hoell et al., 2005; 3 Collinge et al., 1993; 4 Butt & Sultan, 2010; 5 Leishola et al., 2005
3 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
4 Protein Crystallization Nextal.com Non-recombinant
55 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. 3 Collinge et al., 1993,
6 Crude protein porfile Crocus vernus 118kDa 66.2kDa 45.0kDa 35.0kDa 25.0kDa 18.4kDa 14.4kDa Crude Protein profile from corm on SDS-PAGE
7 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 11 Akrem 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) 8
9 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 8000 11 MonodisperseR H = 2.6nmThin sheet 11 Akrem et al., 2011
10 Vapor Diffusion Methods Hanging DropSitting Drop
11 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
13 Protein/Salt Crystals VIS-microscopeUV-microscope1mm size approx. Best is to go for diffraction image
14 Diffraction to 3D Diffraction Image 3D Structure Single Crystal X-ray Bombarment Crystallographic Softwares
15 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, UKESRF, FranceDESY, Germany
16 Crystal Mounting Nylon loops to fish out crystals Goniohead X-ray gun Cryonozzle Microscope Beamstop Detectors
17 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
18 X-ray diffraction Data Space groupC2 Unit-cell parameters (Å,°)a = 172.3, b = 37.1, c = 126.4 Å, β = 127 o V M (Å 3 / Da)2.7 Solvent content (%)54.2 Resolution range (Å)25.0 - 2.1 ( 2.2 - 2.1 ) Total Reflections14,0335 (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 crystal 11 Values in parentheses are for the highest resolution shell. *R merge =∑hkl ∑i | Ii (hkl) – | ⁄ ∑hkl∑i Ii (hkl), where is the mean intensity of the observations Ii (hkl) of reﬂection hkl. 11 Akrem et al., 2011
19 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.orgwww.pdb.org Pdbgoodies input page Phase information and Coordinate information
20 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. 13 Henrissat & Bairoch, 1993
21 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.
22 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. 14 Terwisscha van Scheltinga et al., 1996 Sequence Alignment
23 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.
24 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.