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Investigation Into the Structures of a Lectin

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Investigation Into the Structures of a Lectin INVESTIGATION INTO THE STRUCTURES OF A LECTIN FROM TRICHOSANTHES DIOICA AND ONE FROM ERYTHRINA INDICA & BIOPHYSICAL CHARACTERIZATION OF ARACEAE LECTINS FROM SAUROMATUM GUTTATUM AND ARISAEMA TORTUOSUM THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY POORVA DHARKAR DIVISION OF BIOCHEMICAL SCIENCES NATIONAL CHEMICAL LABORATORY PUNE 411 008 INDIA April 2009 CERTIFICATE This is to certify that the work incorporated in the thesis entitled “Investigation into the structures of a lectin from Trichosanthes dioica and one from Erythrina indica & Biophysical characterization of araceae lectins from Sauromatum guttatum and Arisaema tortuosum.” submitted by Ms. Poorva Dharkar was carried out under my supervision. The material obtained from other sources has been duly acknowledged in the thesis. Date Dr. C. G. Suresh (Research Supervisor) Scientist Division of Biochemical Sciences National Chemical Laboratory Pune 411 008 DECLARATION I hereby declare that the thesis entitled “Investigation into the structures of a lectin from Trichosanthes dioica and one from Erythrina indica & Biophysical characterization of araceae lectins from Sauromatum guttatum and Arisaema tortuosum.” submitted by me to the University of Pune for the degree of Doctor of Philosophy is the record of original work carried out by me under the supervision of Dr. C. G. Suresh at the Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008 and has not formed the basis for the award of any degree, diploma, associateship, fellowship, titles in this or any other University or Institution. I further declare that the material obtained from other sources has been duly acknowledged in the thesis. Poorva Dharkar Date Division of Biochemical Sciences National Chemical Laboratory Pune 411008 Dedicated to…….. Mamma Papa-Amma & Aji CONTENTS Certificate Declaration Acknowledgements Abstract………………...………………………………………………………………………...I List of abbreviations……………………………………………………………………………..IX Chapter 1: Introduction 1.1 Genesis of the thesis………………………………………………………………………….1 1.2. Lectins……………………………………………………………………………………….2 1.2.1 Discovery, Definition & Applied aspect…………………………………………...2 1.2.2 Classification of lectins…………………………………………………………...10 1.2.3 Plant lectins as tools in biology and medicine……………………………………36 1.3. Plant lectins with focus on the lectins studied in this research…………………………….38 1.3.1 Type-II RIPs……………………………………………………………………...39 1.3.2 Legume lectins…………………………………………………………………....47 1.3.3 Monocot Mannose Binding Lectins………………………………………………52 1.4 Lectin-carbohydrate interaction…………………………………………………………….56 1.4.1 Galactose-specific lectins…………………………...…………………………….57 1.4.2 E.corallodrndron lectin…………………….……………………………………..58 1.4.3 Jacalin……………………………………………………………………………..60 1.4.4 Ricin………………………………………………………………………………63 1.4.5 Rhizoctonia solani agglutinin (RSA)……………………………………………..64 1.4.6 Galectin-9 from Mouse……………………………………………….…………..65 1.4.7 Galactosyl-binding lectins from the tunicate Didemnum candidum……………...66 1.4.8 C-type Lectins: CEL-I & CEL-III……………………………………...…...……67 1.4.9 Mannose binding plant lectins…………………………………………………....72 Chapter 2: Materials & Methods 2.1 Summary……………………………………………………………………………………74 2.2 Materials…………………………………………………………………………………….75 2.3 Protein purification……………………………………………………………………….....76 2.3.1 Extraction of lectin………………………………………………………………..76 2.3.2 Ammonium sulfate fractionation…………………………………………………77 2.3.3 Gel filtration on Sephadex G-100………………………………………………...77 2.3.4 Affinity chromatography on Guar gum………………………………………......78 2.3.5 Ion exchange chromatography on DEAE Cellulose……………………………...79 2.4 Biochemical and biophysical techniques for protein characterization……………………...80 2.4.1 Biochemical Assay………………………………………………………………..80 2.4.2 Biochemical techniques for protein characterization……………………………..81 2.4.3 Protein Estimation………………………………………………………………...82 2.5 Biophysical techniques for stability studies & Characterizing Tryptophan microenvironment………………………………………………………………………………82 2.5.1 Circular Dichroism measurement………………………………………………...82 2.5.2 Steady-state fluorescence spectroscopy…………………………………………..83 2.5.3 NBS modification………………………………………………………………...84 2.5.4 ANS-binding assay (Hydrophobic dye binding studies)…………………………………………………………………………………………84 2.5.5 Fluorescence Measurement for ligand (Adenine binding studies)……………….85 2.5.6 Tryptophan accessibility: Quenching of fluorescence intensity by small molecular quencher………………………………………………………………………………………...86 2.5.7 Lifetime fluorescence spectroscopy……………………………………………....86 2.6 Crystallographic methods………………………………………………………………….86 2.6.1 Protein crystallization…………………………………………………………….88 2.6.2 Flash cooling of protein crystals….……………………………………………...92 2.6.3 X-Ray diffraction………………………………………………………………...93 2.6.4 Data collection and processing…………………………………………………...94 2.6.5 Sequence alignment.………………………………………………………….......97 2.6.6 Matthew’s number………………………………………………………………..98 2.6.7 Structure determination………………………………………………………......99 2.6.8 Molecular replacement method………………………………………………....102 2.6.9 Refinement……………………………………………………………………....104 2.6.10 Constraints and restraints………………………………………………………105 2.6.11 Max-likelihood refinement…………………………………………………….106 2.6.12 Analysis and validation of structures…………………………………………..107 Chapter 3: Crystallization and X-ray crystallographic studies on a lectin from Trichosanthes dioica. 3.1 Summary…………………………………………………………………………………..109 3.2 Introduction……………………………………………………………………………….109 3.3 Cucurbitaceae lectins & Type II RIPs………………………………………………….....112 3.4 Crystallization of the TDSL by Hanging drop vapor diffusion method…………………..113 3.5 X-Ray diffraction, Data Collection & Processing………………………………………....114 3.6 Alignment of the N-terminal sequence with the sequence of lectins belonging to this family………………………………………………………………………………………….116 3.7 Structure Solution by Molecular Replacement……………………………………………117 3.8 Problems faced due to non availability of the lectin sequence and alignment with lectins from the same genus with Type II RIPs…………………………………………………………….121 3.9 Structural Refinement…………………………………………………………………......123 3.9.1. Model Building and refinement…………………………………………….......124 3.9.2 Refinement of the A chain: The RIP domain…………………………………....125 3.9.3 Refinement of the B chain: Gal/GalNAc binding lectin domain……………......136 3.9.4. Geometry analysis and subsequent methods to improve the geometry of the refined molecule……………………………………………………………………….......128 3.10 Structural details…………………………………………………………………………130 3.10.1. Structure of the A & C chains: The RIP II domain…………………………...130 3.10.2. Structure of the B & D chains: The Lectin domain…………………………...131 3.10.3. Active site characterization: Binding to Adenine…………………………......131 3.10.4. Disulphide bridges and stability of the lectin………………………………….133 3.11 Evolutionary relationship among the Type II RIPs and TDSL………………………......133 3.12 Conclusion……………………………………………………………………………......134 Chapter 4: Biophysical and stability studies on two Trichosanthes lectins. 4.1 Summary………………………………………………………………………………......135 4.2 Introduction……………………………………………………………………………….135 4.3 Trichosanthes lectins…………………………………………………………….………..136 4.4 Trichosanthes dioica and Trichosanthes anguina lectins………………………………....138 4.4.1 Plant properties & Scientific classification…………………………...…………138 4.4.2 Purification & Biochemical characteristics……………………………………...140 4.4.3 Basis for their Biophysical characterizations……………………………………141 4.5 Results & Discussion……………………………………………………………………...142 4.5.1 Fluorimetric measurements………………………………………...…….……...146 4.5.2 Binding of ANS: a fluorimetric analysis…………………………...……………149 4.5. Conclusions……………………………………………………………………………….150 Chapter 5: Crystallization and X-ray crystallographic studies of a Galactose/N-acetyl Galactosamine specific lectin from Erythrina indica 5.1 Summary…………………………………………………………………………………..151 5.2 Introduction……………………………………………………………………………….151 5.3 Legume Lectins……………………………………………………………………………153 5.3.1 The Characteristic legume lectin fold…………………………………………...154 5.3.2 Canonical legume lectin dimer………………………………………………….155 5.4 Erythrina lectins…………………………………………………………………………...156 5.4.1. Erythina lectins: E.corallodendron, E.cristalgalli & E.indica……………….....156 5.4.2. Erythrina indica seed lectin…………………………………………………….158 5.5. Dialyzing the purified lectin to remove sugars and salts…………………………………159 5.6. Crystallization of EiSL by Hanging-drop vapour-diffusion method……………………..159 5.7. X-Ray diffraction, Data Collection & Processing………………………………………...161 5.8. Alignment of the N-terminal sequence with the sequence of lectins from the same genus…………………………………………………………………………………………..161 5.9 Structure Solution by Molecular Replacement using E.corL coordinates………………...163 5.10 Structure Refinement…………………………………………………………………….165 5.10.1. Model Building and refinement……………………………………………….165 5.10.2. Modelling the lactose molecule in the binding cleft…………………………..166 5.10.3 The N-linked sugars (the heptasachharide and other oligosaccharides)….........166 5.10.4 Placing the metal ions: Ca++ and Mn++ in the coordination sites………….…168 5.10.5 B-Factor Refinement…………………………………………………………..168 5.10.6 Adding solvent molecules to the refined structure………………………….....168 5.10.7 Ramachandran Plot and Procheck analysis of the final model………………...169 5.11. Structural details………………………………………………………………………...173 5.11.1. The subunit structure: Structure of the A & B chains………………………...173 5.11.2. Active site (Architecture) characterization:
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