Purification, characterization and proteomics analysis of important plant proteins from family Brassicaceae
BY BINISH KHALIQ M.Phil. (BZU) A Thesis submitted in partial fulfillment of the requirements for the award of the degree of DOCTOR OF PHILOSOPHY In BOTANY
INSTITUTE OF PURE & APPLIED BIOLOGY
BAHAUDDIN ZAKARIYA UNIVERSITY
MULTAN
2018
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DECLERATION
I Binish Khaliq assert that this is my own work and has not been submitted in any form for another degree at any University. Information consequent from published and unpublished literature of other workers has been acknowledged in the text and a list of references is given.
Binish Khaliq
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Certificate
It is also certify that the research work described in this PhD dissertation entitled ―Purification, characterization and proteomics analysis of important plant proteins from family Brassicaceae‖ is the original work of the author and has been carried out under my direct supervision. I have gone through all its data, contents and results reported in the thesis. I also certify that the thesis has been developed under my supervision according to the prescribed format and I endorse its evaluation for the award of PhD degree in accordance with the prescribed procedure of the university. I further certify that the material included in the thesis has not been used partially or fully, in any thesis already submitted or is in the process of submission in partial or complete fulfillment of the award of any other degree from any institution.
Name Signature
Dr. Ahmed Akrem (Supervisor)
Professor Dr. Muhammad Naeem (Director, IP & AB)
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ACKNOWLEDGEMENTS
I bow my head before the ALMIGHTY ALLAH, who blessed me the ability to complete this research work. I am also praiseful to the last holy prophet HAZRAT
who is the torch bearer of knowledge & guidance for all (ﷺ) MUHAMMAD humanity.
I am deeply appreciative to my research supervisor, Dr. Ahmed Akrem, Assistant professor of Botany, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan for his valuable guidance and dedicated time. More importantly, his constant encouragement, kindness, scientific and technical guidance were major factors that helped me to continue my whole research work and complete my thesis. It was a wonderful experience to work under his supervision. His inspiring guidance thought out the course of this study and especially for introducing me into the field of "Protein Crystallography and Structural Biology".
I am thankful to Professor Dr. Muhammad Naeem, Director; Institute of Pure & Applied Biology, Bahauddin Zakariya University, Multan, for his encouraging attitude and providing necessary research facilities.
I am also thankful to Professor Dr. Dr. Christian Betzel, Institute for Biochemistry and Molecular Biology, University of Hamburg, Germany for helping present research work and for his technical guidance in the field of Protein Crystallography and Structural Biology and providing me with an opportunity to study in this field and in his group. I also convey my thanks to Dr. Friedrich Buck, Universitäts-Klinikum Hamburg-Eppendorf (UKE), Germany, who helped me a lot for the N-terminal sequencing and Mass Spectrometry results which greatly facilitated my progress. I would like to thank Dr. Sven Falke; Institute for Biochemistry and Molecular Biology, University of Hamburg, Germany, for his guidance in learning how to analyze SAXS data.
I also want to extend my words of gratitude to Professor Dr. Seema Mahmood for her valuable support during the course of my PhD work. Additionally, I should also acknowledge her sincere guidance during different times or research. I would also like to send my regards and gratitude to Dr. Muhammad Ibrahim; Department of Biosciences,
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Comsats Institute of Information Technology, Sahiwal Campus, for helping me in getting MALDI-TOF data. I am also thankful to Dr. Qamar Saeed, Department of Entomology, Bahauddin Zakariya University, Multan for helping me in conducting insecticidal assays.
I send all my respect and gratitude to the Higher Education Commission (HEC), Islamabad, Pakistan for the financial support as Indigenous scholarship program, International Research Supportive Initiative Program (IRSIP) in order to work in University of Hamburg, Germany as a part of PhD program.
Finally, I would like to thank all my colleagues who have been in the labs throughout my years of study for providing me with help and support and for being good companions during difficult working times. I am highly obliged to my respectable parents from the core of my heart for being so kind for enriching my academics and for their fruitful prayers for my success. Lastly, I am thankful to my loving siblings Shaban, Sheraz, Bushra, Sehrish & Sidra for their all-time best wishes for my studies.
Binish Khaliq
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CONTENTS
Chapter No. Titles Page No. - ACKNOWLEDGEMENTS VI - CONTENTS VIII - LIST OF FIGURES XIII - LIST OF TABLES XX - LIST OF ABBREVIATIONS XXII - LIST OF PHYSICAL UNITS XXIII LIST OF AMINO ACID SYMBOLS XXIV ABSTRACT XXV 1 INTRODUCTION 1 1.1.1 Brassica nigra (Kali Rai) 3 1.1.2 Eruca sativa (Rocket Salad) 4 1.1.3 Sisymbrium irio (London rocket) 4 1.2 Seed proteins 5 1.2.1 Cruciferins (11/12S Globulins) 6 1.2.2 Ubiquitins 7 1.2.3 Napins (2S Albumin) 8 1.2.4 Thionins 10 1.3 Losses due to pathogen attack 10 1.4 Aims and Objectives 11 2 MATERIALS AND METHODS 12 2.0 Screening of Brassicaceae species 13 2.1 Preparation of seed crude extracts 14 2.2 Protein quantification 14 2.2.1 Bradford reagent 14 2.2.2 Nanodrop 2000c 14 2.3 Gel electrophoresis 15 2.3.1 One dimensional SDS-PAGE 15 2.3.2 Two dimensional (IEF/SDS-PAGE) 15 2.4 Protein identifications 15
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2.4.1 Electroblotting 15 2.4.2 N-Terminal amino acid sequencing 16 2.4.3 Mass-spectrometry of proteins 16 2.4.3.1 MALDI-TOF/TOF Mass-spectrometry 16 2.4.3.2 LC–MS/MS Mass spectrometry 16 2.5 Protein purification 17 2.5.1 Fast Protein Liquid Chromatography (FPLC) 17 2.6 Structural characterization 17 2.6.1 Circular Dichroism (CD) spectroscopy 17 2.6.2 Dynamic Light Scattering (DLS) measurements 18 2.6.3 Small Angle X-ray Scattering (SAXS) 18 2.7 Crystallization of proteins 19 2.7.1 Pre-Crystallization test 19 2.7.2 Robotic screening of crystallization conditions 20 2.7.3 Optimization of initial crystallization conditions 20 2.7.4 Confirmation of protein crystals 21 2.7.5 Data collection and processing 21 2.8 In-Silico analysis 21 2.9 Bioactivity assays 22 2.9.1 Cell cytotoxicity assay 22 2.9.2 Antifungal assay 23 2.9.2.1 Disc Diffusion method 23 2.9.2.2 96-well microtiter plate 23 2.9.3 Entomotoxicity assay 24 2.9.3.1 Statistical analysis 25 2.10 Software and Bioinformatics tools 25 2.10.1 ProtParam 25 2.10.2 Multiple sequence alignment 25 2.10.3 BOXSHADE server 25 2.10.4 ATSAS 25 2.10.5 PDBsum 26
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2.10.6 PROCHECK 26 2.10.7 SWISS-MODEL server 26 2.10.8 Quick 2D server 26 2.10.9 PyMOL 26 2.10.10 Chimera 26 3 RESULTS 27 3.1 Initial screening 28 3.2 Identification and characterization of 12S globulins 30 Extraction and identification of Cruciferin from B. 3.2.1 30 nigra 3.2.1.1 Purification of Cruciferin 31 3.2.1.2 2D Electrophoresis (IEF/SDS-PAGE) of Cruciferin 33 3.2.1.3 Secondary structure determination of Cruciferin 34 3.2.1.4 Dynamic Light Scattering analysis 35 3.2.15 Diffraction data analysis 36 3.2.16 Analysis of SAXS data 37 3.2.2 Identification of Eruca sativa Cruciferin (EsC) 41 3.2.2.1 Purification of EsC 43 3.2.2.2 Secondary structure determination of EsC 44 3.2.2.3 Dynamic Light Scattering analysis of EsC 45 3.2.2.4 Crystallization and Diffraction data analysis of EsC 46 3.2.2.5 SAXS data analysis of EsC 47 3.3 Ubiquitin 50 Protein purification, identification and amino acid 3.3.1 50 sequencing 3.3.2 Characterization in solution of SiU 53 3.3.2.1 CD spectroscopy 53 3.3.2.2 DLS measurements 54 3.3.2.3 In-Silico analysis of SiU 55 3.3.2.4 Quality of the 3D-structure model 57 Structure comparison of SiU to Human Erythrocytic 3.3.2.5 58 Ubiquitin (heu)
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3.4 Napin 61 3.4.1 Extraction and identification of (BnN) 61 3.4.2 Purification of (BnN) 62 3.4.3 Characterization in solution 63 3.4.3.1 CD spectroscopy 63 3.4.3.2 DLS measurement of BnN solution 64 3.4.3.3 Effect of temperature on BnN monodispersity 65 3.4.3.4 Effect of 20 ºC on BnN stability 65 3.4.4 Crystallization of BnN 68 3.4.5 Bioactivity assays analysis 69 3.4.5.1 BnN antifungal activity 69 3.4.5.2 Entomotoxic assessment 70 3.5 Thionin 71 3.5.1 Extraction and identification of pEsT 71 3.5.2 Purification of pEsT 73 3.5.3 Characterization in solution 74 3.5.3.1 Secondary structure determination of pEsT 74 3.5.3.2 Dynamic Light Scattering analysis of pEsT 75 3.5.3.3 Effect of temperature on pEsT stability at 4 ºC 76 3.5.3.4 Effect of temperature on pEsT stability at 20 ºC 76 3.5.3.5 Crystallization of pEsT 79
3.5.3.6 Assessment of tertiary and quaternary structure 79
3.5.4 Bioactivity assays analysis 81 3.5.4.1 Cell cytotoxicity assessment of pEsT 81 Antifungal activity of pEsT by (Disc Diffusion 3.5.4.2.1 82 Method) Antifungal activity of pEsT by (96-well microtiter 3.5.4.2.2 83 plate) 3.5.4.3 Entomotoxic assessment of pEsT 85 4 DISCUSSION 87 4.1 Brassica nigra Cruciferin (BnC) 88
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Conclusions 94 4.2 Eruca sativa Cruciferin (EsC) 95 Conclusions 97 4.3 Sisymbrium irio Ubiquitin (SiU) 98 Conclusions 100 4.4 Brassica nigra Napin (BnN) 101 Conclusions 103 4.5 Precursor Eruca sativa Thionin (pEsT) 104 Conclusions 107 5.0 REFERENCES 109 PUBLICATIONS 135 SAXS and other spectroscopic analysis of 12S 1 cruciferin isolated from the seeds of Brassica nigra (Published) Characterization and In-Silico Studies on Ubiquitin 2 protein from seeds of Sisymbrium irio (Published) Structural characterization of 12S Cruciferin from 3 Eruca sativa in solution using small angle X-ray scattering (SAXS) (Submitted)
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LIST OF FIGURES
Sr. No. Titles Page No. Fig. 1. Pipetting robot for putting different crystallization conditions 1 20 (Zinsser Analytical GmBH, Frankfurt, Germany). Fig. 2. SDS-PAGE showing crude protein profiles of twenty different plant species. Crude extracts were prepared in Tris buffer (100 mM, pH 7.5) and were loaded on SDS-PAGE under non-reduced condition.
Lane 1: (Brassica nigra), 2 (Eruca sativa), 3 (Brassica napus), Ml (Protein ledder), 5 (Sisymbrium irio), 6 (Lepidium sativum), 7 2 (Brassica juncea), 8 (Brassica rapa), 9 (Sinapis alba), 10 (Sisymbrium 29 loeselii), 11 (Brassica campestris), 12 (Lepidium capitatum), 13
(Raphanus raphanistrum), M2 (Protein ledder), 15 (Nasturtium microphyllum), 16 (Iberis amara), 17 (Lobularia maritima ), 18
(Brassica oleracea), 19 (Iberis umbellata), M3 (Protein ledder), 21 (Iberis gibraltarica). Fig. 3. Seeds of three selected Brassicaceae species. (A) Brassica 3 30 nigra (B) Eruca sativa (C) Sisymbrium irio. Fig. 4. (A) Gel filtration chromatogram of the pure Cruciferin. (B) SDS-PAGE is showing the different forms of the 50 kDa monomer 4 32 (L3) e.g., L1 and L2 are indicating the trimeric form while L4 and L5 are showing reduced acidic (α) and basic (β) polypeptides. Fig. 5. Two-dimensional electrophoretic separation of Cruciferin 5 subunits showing α (red box) and β-polypeptides (yellow box) into 33 their respective isoforms. Fig. 6. (A) Circular Dichroism (CD) spectroscopy showing α-helical and dominating β-sheeted conformation. (B) Topology diagram of B. 6 34 napus 11S globulins (PDB ID: 3KGL) illustrating the dominating anti- parallel β-strands. Fig. 7. (A) Dynamic light scattering (DLS) measurement showing 7 polydisperse pure Cruciferin solution in phosphate buffer (pH 6.5). (B) 35 A monodisperse solution dissolved in Tris buffer (pH 7.5). (C)
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Histogram showing the hydrodynamic radius RH (5.8 ± 0.1 nm) confirming the trimeric status. Fig. 8. Crystals of purified 12S globulin/Cruciferin of Brassica nigra. 8 The largest dimension of crystal is approximately 100 µm. Scale bar, 36 0.05 mm. Fig. 9. SAXS analysis and modeling of BnC. (A) Processed solution scattering pattern of B. nigra Cruciferin (blue) in arbitrary intensity units and the fit with B. napus 11S globulin (ProCruciferin; PDB ID: 9 38 3KGL) (red) as evaluated by CRYSOL (χ2 = 1.34). (B) Kratky plot of the scattering data verifying an overall compact and rigid folding. (C) Corresponding distance distribution functions p(r).
Fig. 10. SAXS analysis and modeling of BnC. Ab initio model of 10 Cruciferin with P3 symmetry (grey spheres) and B. napus 11S globulin 39 (PDB ID: 3KGL) were superimposed. Fig. 11. Sequence alignments of EsC to the others plant Cruciferins; Cruciferin (CRU1) from Raphnus sativus and Cruciferin (CRU3) from Brassica napus, indicating a high level of residual identity (63 %). 11 Identically conserved sequences are represented by Asterisks (*) under 42 sequences while conserved substitutions and semi-conserved substitutions are represented by double dots (:) and single dots (.) respectively. Fig. 12. Purification and SDS-PAGE analysis of EsC (A) A highly purified EsC was obtained after gel filtration (Hi-Load 16/60 Superdex 200 column) chromatography. (B) 12 % SDS-PAGE of the purified Cruciferin from E. sativa fractions. Lane M contains molecular weight 12 43 standards. Lane 1; purified Cruciferin (50 kDa approximately highlighted in red box) without mercaptoethanol; Lane 2: under reduced form in presence of mercaptoethanol showing two bands of 30 and 22 kDa also known as α and β-polypeptides respectively. Fig. 13. Averaged far-UV CD spectrum of seed EsC. Although CD spectra is indicating the presence of both helix and sheets, however, 13 44 the major secondary structure is β-sheeted followed by nonstructural or loop areas.
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Fig. 14. Dynamic light scattering (DLS) measurement of E. sativa native Cruciferin (EsC). (A) is showing the monodisperse nature of Cruciferin solution at a concentration of 41 mg/ml. (B) is showing a 14 45 hydrodynamic radius (RH) of 5.5 ± 0.36 nm confirming the trimeric nature with molecular weight of approximately 170 kDa for the real molecule. Fig. 15. In situ microscopic analysis of EsC crystals with and without UV light. (A) is showing the small sized Cruciferin crystals (0.1 × 0.05 15 46 × 0.05 mm) under visible microscope while (B) is showing the same crystals producing fluorescence under UV light. Fig. 16. SAXS analysis and modeling of EsC. (A) Processed solution scattering pattern of EsC (green) in arbitrary intensity units and the fit with B. napus 11S globulin (ProCruciferin; PDB ID: 3KGL) (blue) as 16 48 evaluated by CRYSOL (χ2 = 2.0). (B) Kratky plot of the scattering data verifying an overall compact and rigid folding. (C) Corresponding distance distribution functions p(r). Fig. 17. SAXS analysis and corresponding ab initio model of EsC with P3 symmetry (grey spheres) and 3KGL have been superimposed. It 17 49 indicated that native EsC molecules exist in trimeric form possessing a molecular weight of approximately 180 kDa. Fig. 18. Extraction, purification and isoelectric focusing of SiU. (A) SDS-PAGE analysis of S. irio crude extract at pH 3.0 in lane L, (B) Size-exclusion chromatogram obtained from Superdex 200 column, 18 (C) SDS-PAGE analysis of SiU protein, comprising lane M for 52 molecular weight standard, lane L1 for non-reducing condition and lane L2 for reducing condition. (D) Isoelectric focusing (IEF) of purified SiU showing single band on strip with the alkaline pI of 9.2. Fig. 19. Circular dichroism of SiU showed Far-UV CD spectrum that 19 53 consist of high content of 39.3 % β-sheet. Fig. 20. Dynamic light scattering of Ubiquitin (A) DLS and (B) the 20 54 hydrodynamic radius (RH) of purified SiU. Fig. 21. Multiple sequence alignment of Ubiquitin proteins from 21 56 different species present in UniProtKB database. Black background
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showed overall conserved regions and grey background also revealed conserved regions but not obtained through LC–MS/MS in Siu sequence, - represents gaps whereas * indicates exactly identical residues. Red boxes show conserved changes while green boxes represent non-conserved changes. 22 Fig. 22. Ramachandran plot of the modeled SiU structure. 57 Fig. 23. Structural features of SiU. (A) Human erythrocytic Ubiquitin (PDB-ID: 1UBQ) and (B) S. irio Ubiquitin (SiU). Structural motifs 23 60 include F4 patch yellow, TEK box orchid, I36 patch salmon, and I44 patch cyan in colors Fig. 24. Purification and SDS-PAGE analysis of BnN from Brassica nigra. (A) is Indicating Size-exclusion chromatogram obtained from Superdex 75 column. (B) 15% SDS-PAGE analysis of B. nigra purified protein (BnN); lane M is showing for molecular weight 24 62 standard, lane L1 is showing the intact 16 kDa BnN under non- reducing condition and lane L2 is showing the reduction of 16 kDa into smaller sub-unit of approximately 10 kDa while smaller sub-unit of 6 kDa is not visible. Fig. 25. CD spectra indicating the secondary conformation of Brassica nigra Napin (BnN). Although BnN is composed of both helices and 25 63 sheets, however, the major secondary structure is α-helix and is compactly folded. Fig. 26. Dynamic light scattering (DLS) measurements of BnN. (A) is confirming a monodisperse solution of BnN at a concentration of 15 26 64 mg/ml. (B) is indicating the hydrodynamic radius (RH) (2.0± 0.1 nm) of purified BnN. Fig. 27. DLS measurements are showing the effect of 4 ºC temperature on the monodispersity of the BnN for three continuous days. A1, B1 and C1 are showing the monodisperse stability of BnN at first, second 27 66 and third day respectively. Similarly, A2, B2 and C2 are showing the corresponding hydrodynamic radii (2.16± 0.3) for three consecutive days with zero fluctuation. 28 Fig. 28. DLS measurements are showing the effect of 20 ºC 67
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temperature on the monodispersity of the BnN for three continuous days. A1, B1 and C1 are showing the monodisperse stability of BnN at first, second and third day respectively. Similarly, A2, B2 and C2 are showing the corresponding hydrodynamic radii (2.16± 0.3) for three consecutive days with zero fluctuation. Fig. 29. In situ microscopic analysis of BnN micro crystals. (A) is showing the small sized Napin crystals (0.035 × 0.02 × 0.018 mm) 29 68 under visible microscope while (B) is showing the zoom in view of the same crystal. Fig. 30. Inhibitory activity of BnN towards Fusarium solani (A) and Fusarium oxysporum (B). [a: Topsin® fungicide (20 µl/disc); b: 30 30 69 µl/disc 20 mM acetate buffer (pH 5.5); c: Diluted BnN protein (15 µg/disc); d: Concentrated BnN protein (30 µg/disc)]. Fig. 31. Sequence homology of Precursor Eruca sativa Thionin (pEsT) 31 to Thi2.4 from Arabidopsis thaliana. Symbol ―X‖ is representing the 72 gaps between the obtained peptides. Fig. 32. Sequence alignments of precursor Eruca sativa Thionin 32 (pEsT) to other plant Thionins. (Thi 2.4) Thionin from Arabidopsis 72 thaliana, (THN) Thionin from Brassica rapa. Fig. 33. (A) Purification of pEsT by calibrated size exclusion chromatography (HiLoad 16/60 Superdex 75) subsequent to ammonium sulfate precipitation. The absorbance at a wavelength of 280 nm is displayed and the absorbance peak of pure Thionin with a 33 retention volume of 80.7 ml corresponding to approximately 20.3 kDa 73 is labeled. (B) SDS-PAGE analysis of pEsT under non-reducing conditions (lane 1) and in the presence of 20 mM DTT (lane 2). Upon addition of DTT the 16 kDa pre-Thionin is split into domains of approximately 11 and 5 kDa respectively. Fig. 34. Far-UV CD spectrum of pEsT from E. sativa as an average of 10 individual spectra in 25 mM phosphate buffer at pH 7.0. Spectra 34 74 indicate that secondary structure of Thionin has major part of α-helix and two distinct minima at around 210 nm and 221 nm. 35 Fig. 35. The Particle size distribution obtained by dynamic light 75
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scattering reveals the monodispersity of the pEsT solution. A
hydrodynamic radius (RH) of 2.5 ± 0.1 nm was determined, which would corresponding to a molecular weight of around 25 kDa, if the particle is globular. Under reducing conditions the hydrodynamic radius decreases to 1.8 ± 0.2 nm. Fig. 36. DLS measurements are showing the effect of 4 ºC temperature on the monodispersity of the pEsT for three continuous days. A1, B1 and C1 are showing the monodisperse stability of pEsT at first, second 36 77 and third day respectively. Similarly, A2, B2 and C2 are showing the corresponding hydrodynamic radii (2.00 ± 0.2) for three consecutive days with zero fluctuation. Fig. 37. DLS measurements are showing the effect of 20 ºC temperature on the monodispersity of the pEsT for three continuous days. A1, B1 and C1 are showing the monodisperse stability of pEsT 37 78 at first, second and third day respectively. Similarly, A2, B2 and C2 are showing the corresponding hydrodynamic radii (2.10 ± 0.3) for three consecutive days with zero fluctuation. Fig. 38. In situ microscopic analysis of pEsT micro crystals. (A) is showing the small sized pEsT crystals (0.025 × 0.02 × 0.022 mm) 38 under visible microscope while (B) is showing the optimization Pact 79 D12 condition in Linbro plate. Again micro crystals were obtained and diffracted to low. Fig. 39. (A) Small-angle X-ray scattering intensity plot of pure pEsT and the corresponding calculated fit curve (red) resembling a single ab initio model as displayed in panel C. The plot shows the dependency of the scattering intensity I on the momentum transfer s. (B) Kratky 39 80 plot of the scattering intensity distribution indicating a compact relatively rigid and compact protein structure (C) Ab initio model calculated by DAMMIF possessing P1 symmetry; the scale bar is 1 nm in length. Fig. 40. Effect of different concentrations of pEsT on Huh-7 cells 40 81 visualized by plotting the cell viability (%) versus concentration (µM). 41 Fig. 41. Inhibitory activity of pEsT towards Fusarium solani (A) and 82
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Fusarium oxysporum (B). [a: Fungicide; b: Water; c: Diluted protein (15 µg/dis); d: Concentrated protein (30 µg/dis)]. Fig. 42. Line chart with the standard deviation from 0h to 120h growth of pEsT. Maximum fungal hypha growth was observed on different concentrations of BSA with 30 µg showing the maximum growth. This is probably, the fungi is consuming the protein for its growth. Among 42 three used concentrations of pEsT, 50 µg showed the maximum 83 inhibition of the fungal growth in comparison to BSA treatments. Significant growth difference could be observed between 30 µg BSA treatment with that of 50 µg pEsT after 96 hours of the recorded time period.
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LIST OF TABLES
Sr. No. Titles Page No.
Table 1. List of different Brassicaceae species used for screening of 1 13 important proteins.
Table 2. List of selected plants and seed proteins that have been 2 identified and characterized among enlisted genera of Brassicaceae 29 family. Table 3. N-terminal sequence comparison showed high homology (82 %) with A. thaliana Cruciferin and similarly with other 11S globulins. 3 Interestingly three different amino acids (highlighted in red) Serine, 31 Asparagine and Glutamic acid were observed among the sequence of B. nigra Cruciferin. 4 Table 4. SAXS data collection and analysis parameters. 40 Table 5. LC-MS/MS spectroscopic produced amino acid sequences of 5 41 EsC. Table 6. LC–MS/MS mass spectroscopic produced amino acid 6 50 sequences of SiU. Table 7. Sequence similarity of identified peptide fragments obtained 7 51 from LC–MS/MS. Table 8. N-terminal sequence comparison showed high homology 8 (78.8%) with R. sativus Napin (2S albumin) and similarly with others 61 Brassicaceae. Table 9. Entomotoxin activity of BnN against different life stages of T. castaneum. Three different Napin concentrations (1, 2 and 3 mg/ml) 9 70 were used to see the insecticidal efficacy. Control was comprised of 20 mM acetate buffer of pH 5.5. Table 10. Random fragmented amino acid sequence of pEsT obtained 10 through MALDI-TOF/TOF mass spectrometry. In total four fragments 71 comprising of 58 residues were obtained. Table 11. Comparison of pEsT protein with Thionins of other plant 11 84 species.
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Table 12. Entomotoxin activity of pEsT against different life stages of T. castaneum. Three different Thionin concentrations (1, 2 and 3 12 86 mg/ml) were used to see the insecticidal efficacy. Control was comprised of 20 mM phosphate buffer, pH 7.0.
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LIST OF ABBREVIATIONS
Abbreviations Augmentations BnC Brassica nigra Cruciferin BnN Brassica nigra Napin BSA Bovine Serum Albumin CCP4i Collaborative Computational Project Number 4 cDNA Complementary DeoxyriboNucleic Acid CV Column Volume DESY Deutsches Elektronen-Synchrotron DLS Dynamic Light Scattering DMEM Dulbecco‘s Modified Eagle Medium DMSO Dimethyl Sulfoxide EMBL European Molecular Biology Laboratory EsC Eruca sativa Cruciferin FPLC Fast Protein Liquid Chromatography MWCO Molecular Weight Cut Off NCBI National Center for Biotechnology Information PAGE Poly Acrylamide Gel Electrophoresis PBS Phosphate Buffered Saline PCT Pre-Crystallization Test PDB Protein Data Bank PEG Polyethylene Glycol pEsT Precursor Eruca sativa Thionin SAXS Small Angle X-ray Scattering SDS Sodium Dodecyl Sulfate SiU Sisymbrium irio Ubiquitin Tris Tris(hydroxymethyl)aminomethane UEA Ulex europaeus Agglutinin UV Ultraviolet
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LIST OF PHYSICAL UNITS
Symbols Augmentations ° Degree °C Degree Celsius µm Micrometer Å Angstrom g Gram K Kelvin kDa Kilo Dalton M Mol mg Milligram min Minute ml Milliliter mm Millimeter mM Milli-molar nl Nano liter nm Nanometer
Rg Radius of Gyration
RH Hydrodynamic Radius Rpm Revolutions per minute λ Wavelength µl Micro liter ms Micro-second χ2 Chi square
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LIST OF AMINO ACID SYMBOLS
Full Name Three Letter Notation Single Letter Notation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
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ABSTRACT
Brassicaceae or Cruciferae is one of the most wide spread economically important plant family comprising of many weed vegetables, condiments and oilseed crops. Pakistan is growing oilseed Brassicaceae species over an area of 272,100 ha with an oil production of 232,000 tons per year. Additionally, very strong protein content (38- 40%) is also present in different genera of this family. Brassicaceae species have a notable economic value due to their nutritionally and medicinally important seed proteins in addition to their oil content. Initially 18 different species from Brassicaceae family were screened in search of important seed proteins. Among the commercially cultivated Brassicaceae species, Brassica nigra, Eruca sativa and Sisymbrium irio have significant amounts of proteins in their seeds. Cruciferins (11/12S globulins) and Napins (2S) are the predominant storage proteins while other proteins like Ubiquitins and Thionins are also important proteins of Brassicaceae seeds that contribute to different properties and functions in comparison to storage proteins. Brassica nigra Cruciferin (BnC): Cruciferins (12S globulins) are seed storage proteins, which are getting attention due to their allergenic and pathogenicity related nature. This study describes the purification and characterization of a trimeric (~ 190 kDa) Cruciferin protein from the seeds of Brassica nigra (L.). Cruciferin was first partially purified by ammonium sulfate precipitation (30 % saturation constant) and further purified by size exclusion chromatography. The N-terminal amino-acid sequence analysis showed 82% sequence homology with Cruciferin from Arabidopsis thaliana. The 50-55 kDa monomeric Cruciferin produced multiple bands of two major molecular weight ranges (α-polypeptides of 28-32 kDa and β-polypeptides of 17-20 kDa) under reduced conditions of SDS-PAGE. The 2D gel electrophoretic analysis showed the further separation of the bands into their isoforms with major pI ranges between 5.7-8.0 (α-polypeptides) and 5.5-8.5 (β-polypeptides). The Dynamic Light Scattering (DLS) showed the monodisperse nature of the Cruciferin with hydrodynamic radius of 5.8 ± 0.1 nm confirming the trimeric nature of the protein. The Circular Dichroism (CD) spectra showed both α-helices and β-sheets in the native conformation of the trimeric protein. The pure Cruciferin protein (40 mg/ml) was successfully crystallized; however, the crystals diffracted only to low resolution data (8Å). Small-Angle X-ray Scattering (SAXS) was applied to gain insights into the three-dimensional structure in solution.
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SAXS showed that the radius of gyration is 4.24 ± 0.2 nm and confirmed the nearly globular shape. The SAXS based ab initio dummy model of B. nigra Cruciferin was compared with 11S globulins (PDB ID: 3KGL) of B. napus which further confirmed a highly similar molecular weight and globular shape indicating a conserved trimerization of B. nigra Cruciferin. The comparison of the scattering patterns of both proteins showed a minimized χ2-value of 1.337 confirming a similar molecular structure. This is the first report describing the purification and characterization of a Cruciferin protein from seeds of B. nigra.
Eruca sativa Cruciferin (EsC): E. sativa Cruciferin (12S globulin) protein was identified and characterized from seeds in trimeric form. The protein was identified by feeding the LC-MS/MS mass spectrometric residual data in UniProtKB online server which indicated 63 % sequence identity with Cruciferins (CRU1) of Raphanus sativus and Brassica napus (CRU3). SDS-PAGE exhibited multiple isoform protein banding pattern of 50-52 kDa monomeric Cruciferin which was separated into α-polypeptides of 30 kDa and β-polypeptides of 20 kDa under reduced conditions. Secondary structure contents of EsC were analyzed by Circular Dichroism spectroscopy which indicated 7.5 % α-helix, 47.6 % β-sheet, 7 % β-turn and 37.9 % random conformation. The monodispersity and stability of EsC for Crystallization was performed through Dynamic Light Scattering (DLS) and hydrodynamic radius of EsC was calculated as 5.5 ± 0.3 nm which further confirmed the trimeric nature of the protein. The pure Cruciferin protein (41 mg/ml) produced rather tiny crystals (0.1 × 0.05 × 0.05 mm) which yielded low resolution diffraction data of 7Å only. SAXS analysis showed that the molecular shape of EsC is globular and oligomeric state of the protein revealed a gyration radius of 4.3± 0.30 nm. The ab initio dummy model of EsC with P3 symmetry was thoroughly compared with 11S globulins (PDB ID: 3KGL) of B. napus which indicated a highly similar molecular weight and more importantly globular shape corresponding to a conserve trimerization of EsC molecule in the solution form. Moreover, the scattering patterns of both proteins showed a minimized χ2-value of 2.0 which further confirms the similar molecular conformation.
Sisymbrium irio Ubiquitin (SiU): Ubiquitin protein was identified and characterized from the seeds of Sisymbrium irio. Results of LC-MS/MS spectrometry revealed that Sisymbrium irio Ubiquitin (SiU) shares around 77 % sequence identity with
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already reported Ubiquitin proteins from Triticum aestivum, Avena sativa, Arabidopsis thaliana and others. SiU revealed a molecular weight of 17 kDa on non-reduced SDS- PAGE while a reduced band of approximately 9 kDa in the presence of β- mercaptoethanol. A pI of 9.2 was calculated by isoelectric focusing measurement. Circular Dichroism (CD) spectroscopic studies showed that the secondary structure of Siu comprised of about 6.5 % α-helix, 39.3 % β-sheet, 0.7 % turn, 53.5 % random coil which indicates that it is mostly β-sheeted. Similarly, a hydrodynamic radius (RH) of 2.44 nm was calculated by Dynamic Light Scattering (DLS) for monodispersive SiU which further infers the presence of a dimeric status of 17 kDa native protein in solution. Multiple sequence alignment of 20 different Ubiquitin sequences revealed that Ubiquitin sequence is well conserved in nature with very few amino acid differences, e.g., R42K, Q49D, and Q62D. Homology modeling of SiU (11-28, and 42-76) residues suggested the overall conserved secondary and tertiary conformation and other structural motifs.
Brassica nigra Napin (BnN): A 16 kDa BnN was identified by N-terminal amino acid sequencing which showed 78.8 % homology to that of Napin (2S albumin) from Raphanus sativus and 71.4 % identity to Napin-3 and Napin proteins from Brassica napus and Brassica campestris respectively. BnN was made highly purified by optimizing a combination of Cation exchange and size-exclusion chromatography. Purified BnN showed a typical banding pattern on 15 % SDS-PAGE exhibiting approximately 16 kDa molecular weight under non-reduced condition while a visible band of 10 kDa in the presence of β-mercaptoethanol confirming the presence of inter-chain disulfide linkage between two chains. However, the other cleaved band of approximately 5-6 kDa is not visible on gels. The secondary structure of BnN was studied by Circular Dichroism (CD) spectroscopy and it showed that BnN consists of about 38 % α-helix, 20 % β-sheet and 11 % β-turns. DLS data indicated the monodispersity and stability of BnN in 20 mM acetate buffer of pH 5.0. DLS data recording also indicated the zero effect of two temperatures (4 and 20ºC) and BnN monomeric conformation remained stable at both temperatures.
Moreover, a hydrodynamic radius (RH) of 1.99 nm was calculated which confers the monomeric nature of the 16 kDa BnN in solution form. Purified BnN (15 mg/ml) produces small size diamond shape crystals (0.035 × 0.02 × 0.02 mm) in 0.1 M Sodium citrate, pH 5.5, 20 % w/v PEG 3k condition. However, crystals diffracted to poor resolution (10Å) and as a result no structure was solved. Nevertheless, BnN exhibited strong antifungal activity against phytopathogenic Fusarium solanii and Fusarium
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oxysporum at a concentration of 30 µg per disc. Similarly, BnN showed good insecticidal activity against T. castaneum larval growth at a concentration of 3 mg/ml. Similarly, total number of pupae and adults were also significantly reduced in comparison to control experiment. Indeed comparison of treatments to control; indicated the highest mortality due to BnN treatment for all three populations (average larval, pupal and adult) in comparison to control.
Precursor Eruca sativa Thionin (pEsT): Another 16 kDa Precursor Eruca sativa Thionin (pEsT) was identified by MALDI-TOF/TOF mass spectrometry. The fragmented amino acid sequence was BLAST in UniProtKB online server and result showed a 100 % sequence identity with Thionin 2.4 of Arabidopsis thaliana. Purified pEsT showed a compact 16 kDa protein band on 15 % SDS-PAGE under non-reduced condition while in the presence of β-mercaptoethanol; it produces two bands of 11 and 5 kDa. Secondary structure of pEsT was calculated by using CD spectroscopy and spectra indicated the 38 % α-helix, 9 % β-sheet, 19 % turn and a higher content of 34 % which was not compactly folded (random). Monodispersity and stability of pEsT was checked by DLS for three consecutive days at two different temperatures (4 and 20°C). Poor quality diffraction data were obtained from rectangular shape pEsT crystals. An elongated shape dummy model of pEsT was calculated by SAXS scattering pattern (Rg of 1.96 ± 0.1 nm). The ab initio model calculated by DAMMIF used P1 symmetry and a volume of 31100 nm3 which corresponded to approximate 15.5 kDa molecular weight of protein. pEsT showed the cytotoxity against the hepatic cell line Huh7 and LC50 was obtained at ≥ 12.5 µM. Antifungal activity of pEsT was checked against Fusarium solanii and Fusarium oxysporum by Disc diffusion method while 96 well-microtiter plate methodology was used against Fusarium graminearum. pEsT significantly inhibited the growth of these pathogenic fungi. pEsT protein showed strong Entomotoxic activity against different life stages of T. castaneum growth.
.
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INTRODUCTION
Chapter 1 Introduction
Family Brassicaceae or Cruciferae is one of the most wide spread plant kingdom. Brassicaceae species are grown worldwide for food, feed and oil purposes. There are 370 genera and 3,200 species of plant family Brassicaceae (Cruciferae). Brassicaceae is an economically important plant family with wide spectrum agronomic traits ( Perera et al., 2016; Love et al., 2005). Many vegetable weeds, condiments, oilseeds and economically important crops belong to Brassicaceae. Some species have been used as vegetables, in the Neolithic age, (Downey & Robbelen, 1989). The vegetables of Brassicaceae are Brassica. napus (Kale), Brassica oleracea (Cauliflower and Cabbage) and Raphanus sativus (Radish) (Downey & Robbelen, 1989). Some dominant weed species are Raphanus raphanistrum, Brassica rapa, Sisymbrium irio and Lepidium sativum. Different plant parts like leaves, inflorescences, roots, bulbs, stems and seeds have been recognized as part of edible food since historic times.
Brassicaceae species are the earliest domesticated plants. Major species of Brassicaceae that are mainly grown as oilseeds include Brassica juncea, Brassica napus and Brassica campestris. Brassicaceae oilseeds are for the most part developed for good to eat and modern oil generation purposes. Altogether, the oilseeds of Brassicaceae contain 9 % of the world's consumable vegetable oil provide. Oilseeds of Brassicaceae are 3rd important critical source of palatable oil after palm and soybean according to food and agriculture organization (FAO, 2009). The Residual canola dinner after oil extraction is an appropriate source of protein for creature nourish and mechanical approach (Newkirk, 2009). In the early 16th century, rapeseed was commercially grown. In Europe, rapeseed oil was used in steam engines and lamp oil as lubricants. The crops of Brassicaceae oilseed are natively present in the temperate regions of the world. The low temperature and moisture condition are favorable for survival and production of seeds of these plants and has made them adjustable to cool environment, high assessment and subtropical farming. Oilseed rape (Brassica napus) and turnip rape (Brassica rapa) are mainly cultivated in temperate parts of North America, China, tropical and semi-tropical regions such as of Europe and Asia. Brassicaceae oilseed crops have significant economic importance in Western Canada. B. napus, B. rapa and recently B. juncea have low glucosinolate and low erucic acid. Varieties of Brassica juncea that fit glucosinolates and canola‘s oil profile Canola Quality Mustard; (CQM) have been developed. Brassica juncea has well-adjusted against the semi-arid areas of Western Canada and is defiance to
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Chapter 1 Introduction black-leg disease and fatty acid profile is near to close related to Brassica napus (Potts et al., 1999).
The Brassica juncea commonly known as Indian sarson or Rai is mainly cultivated in Pakistan, India and Bangladesh. The Brassica carinata (Ethiopian or Abyssinian sarson) has been originated in the sub-Saharan areas of the world (Downey & Robbelen, 1989). Brassica juncea and S. alba are cultivated for the condiment mustard market. In Saskatchewan, they are economically important crops. Mustard plants have an uncommon adjustment to Western atmosphere and Canadian soil. They are heat, dry spell tolerant and impervious to various diseases. Cruciferous vegetables are a good source of nutrients and phytochemicals, including fiber, folate, chlorophyll and carotenoids. Cruciferous vegetables have a high quantity unique glucosinolates and sulfur containing compounds. These compounds have spicy (bitter) taste and pungent aroma (Drewnowski & Gomez, 2000). High intake of cruciferous vegetables may reduce the risk of lung, colorectal, breast and prostate cancer in humans because cruciferous vegetables are a good source of glucosinolates (secondary metabolites) and their products, including isothiocyanates and indoles (Bones & Rossiter, 2006). Epidemiological studies show that human cancer risk may decrease as intake of indoles and isothiocyanates through Brassicaceae vegetable (Verhoeven et al., 1996).
1.1.1 Brassica nigra (Kali Rai)
Brassica genus of family Brassicaceae possesses about 150 species. Brassica nigra is an annual condiment weedy crop (Muluye et al., 2015). Brassica nigra is used for various aspects of food, medicine, vegetable and industrial products throughout the world. Brassica nigra plant is enriched with many pharmacologically active phytochemicals, such as flavonoids, alkaloids terpenoids and tannins (Shafaghat, 2010; Tomar & Shrivastava, 2014) and is reported to show antiplasmodial activity (Bero et al., 2009). The seeds of Brassica nigra have been used as an appetite stimulant, tonic, digestive, laxatives, antiseptics and curative for tumours and carcinoma (Amri, 2014). Similarly, these seeds are used as medicine against colds, abscesses, rheumatism, headaches, neuralgia, edema, epilepsy, alopecia, snakebite, hiccup, toothache, pneumonia (Muluye et al., 2015) and malaria (Ghafari et al., 2013). The seed extracts of Brassica nigra exhibited potent antiepileptic activity (Praveen et al., 2013) which might be
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Chapter 1 Introduction important for the management of cerebral malaria. The seeds of Brassica nigra are a good source of protein e.g., storage proteins are accumulated in large amounts in plant seeds during their development. According to Wan et al. (2007) they are used as the main nitrogen source for seed development and early seedling growth.
1.1.2 Eruca sativa (Rocket Salad)
Eruca sativa (Rocket Salad) locally known as Taramira is an annual herb and is cultivated in different parts of the Indo-Pak subcontinent as well as in the Middle East (Martínez-Sánchez et al., 2006). Eruca sativa is a minor oil crop; widely used as culinary and medicines as treatment for many diseases. There is only scattered information present about phytochemistry and bioactivity of this oily plants (Flanders & Abdulkarim, 1985). The regular consumption of Eruca sativa has been associated with the prevention of cardiovascular diseases and reduction in cancer (Fuentes et al., 2014; Higdon et al., 2007). It is known as diuretic and anti-inflammatory food (Yehuda et al., 2012). Eruca seeds possess various proteins, glucosinolates, vitamins A and C, flavonoids, erucic acid and high oil contents (Bell & Wagstaff, 2014; Kim & Ishii, 2006). It is most commonly utilized as animal feed in Asia, specifically in India and Pakistan. Rocket plants have also greater quantity of polyglycosylated flavonol compounds that have inferred numerous valuable health effects in humans and other animals. Specifically they have effects on the cardiovascular health and gastrointestinal tract (Björkman et al., 2011; Traka & Mithen, 2011).
1.1.3 Sisymbrium irio (London rocket)
Sisymbrium species is one of Cruciferae members which have many uses in folk medicine in a treatment of inflammation and rheumatoid(Vohora et al., 1980). Sisymbrium irio (London rocket) locally known as khub kalana, is an annual herb and it is present in family Brassicaceae. It is cultivated in different regions of the Indo-Pak subcontinent and in the Middle East. Sisymbrium irio is a minor oil plant widely utilized as culinary and for medicines as remedies for different diseases. Sisymbrium irio L. is widely distributed in Saudia Arabia (Al-Jaber, 2011). The seeds were used as an expectorant and as a febrifuge (Ghazanfar, 1994). This genus was found to contain flavonoids (Khan et al., 1991), alkaloids, anthraquinones (Arayno & Zafor, 1983), oils, steroids (Soulier, 1994) and glycosides (Krets et al., 1987). Some workers mentioned that Page 4
Chapter 1 Introduction
Sisymbrium irio has antipyretic, analgesic and anti-microbial activities (Vohora et al., 1980).
1.2 Seed proteins
Brassicaceae species have a notable economic value due to their medicinal, nutritional, biocontrol, bio-industrial and plant succession characteristics (Montesano et al., 2003). Mustard and oilseed rape have been used for medicinal purposes, according to Roman Greek and Chinese writings in 2000 BC to 5000 BC and Indian Sanskrit writing in 1500 to 2000 BC. Brassicaceae seeds are rich in oil and have 20 to 35% protein in a significant amount of seed dry weight. In Brassicaceae, all protein content is seed cotyledon or whole seeds itself. In Brassicaceae oilseed, the main nitrogenous compounds are seed storage proteins (SSPs). Proteins are mainly stored in seed embryo and cotyledons in the form of protein storage vacuoles (PSVs) (Jiang et al., 2001). During seed germination, protein start to accumulate and the stored protein released the N and S that required during seed germination (Ashton, 1976; Müntz, 1998). The lytic enzymes phytates and phosphorous-rich compounds (make insoluble crystals) are also stored in PSVs in addition to seed storage proteins. During the seed germination, lytic enzymes help to breakdown the SSP (Herman & Larkins, 1999). The PSVs are spherical organelles present inside seeds of Brassicaceae species ranging from 1.5 to 8 μm in diameter (Ashton, 1976).
In the family Brassicaceae, there are two types of SSPs exist. The seed storage proteins like 11/12S Globulins which are known as Cruciferins. The cruciferins are present in abundant quantity than 2S albumin napin which have 12-16 kDa molecular weight (Crouch & Sussex, 1981; Lönnerdal & Janson, 1972). Since the production of Cruciferins and Napins involve the expression of multigene, therefore a high-level of polymorphism exists between these proteins and therefore no adequate method has been succeed to quantify each type of protein and isoforms of protein in Brassicaceae oilseeds.
Brassicaceae oilseed has fulfilled the human nutritional need of protein quantities (Sims, 1971). Additionally, seed meals contain glucosinolates; phytic acid, phenolic compounds and fibers suitable for food and feed need (Bell, 1993; Sims, 1971). Brassica plants have the myrosinase enzyme that are abundantly exist in myrosin cells (Bones et al., 1991; Kissen et al., 2009). According to Falk et al. (1995), there are three gene
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Chapter 1 Introduction families of myrosinases (MA, MB and MC) that are present among different species of genus Brassica. Myrosinases are present in dimeric form and glycosylated proteins with 62 to 75 kDa molecular weight (Bones & Rossiter, 1996). Myrosinases of MA family occur as a free dimers approximately 140 kDa, while myrosinases of MB and MC families exist in the form of high molecular complexes around 200 to 1000 with myrosinase associated proteins (MyAp) and myrosinase binding proteins (MBP) (Rask et al., 2000).
1.2.1 Cruciferins (11/12S Globulins)
Cruciferin belongs to cupin protein super family. It is present in large quantity of the storage proteins in seeds of Brassicaceae species. The cruciferin is about 60% of the total seed storage proteins in mature Brassicaceae seeds. It has molecular mass ranges from 300-310 to 350-355 kDa (Schwenke et al., 1981; Sjodahl et al., 1991; Crouch & Sussex, 1981). Malabat and his co-workers reported that cruciferin protein may vary from 32-52% of total proteins in the European rapeseed species (Malabat et al., 2003).
Bilodeau et al. (1994) reported that cruciferins are expressed by 9 to 12 genes in B. napus (heterogeneous), while only 3 genes are involved in Arabidopsis. In B. napus; there are five main groups of cruciferin subunit such as CRU1, CRU2, CRU3, CRU2/3, and CRU4 (Rödin & Rask, 1990). The molecular weight of each subunit is approximately 50 kDa which in turn is composed of α-polypeptide (acidic) of 30 kDa and β-polypeptide (basic) of 20-25 kDa, these polypeptides are linked by disulfide bonds (DeLisle & Crouch, 1989). B. napus cruciferin (50 kDa) is composed of 443 to 486 amino acids. Isoelectric point (pI)of cruciferin is approximately 7.25 (Dalgalarrondo et al., 1986). Cruciferin can produce many polypeptide bands on SDS-PAGE under reducing conditions in the region of 30 kDa and 20 kDa because of isoform micro heterogeneity (Delseny & Raynal, 1999).
Aluko and Katepa (2005) reported that meals of S. alba and Brassica juncea produced polypeptide bands in the range of 50, 55 and 135 kDa on SDS-PAGE in the absence of β-marcaptoethanol while 50, 55, and 135 kDa bands disappeared under reducing conditions and low molecular weight polypeptide were created which indicated the existence of disulphide bonds in the mother proteins (Aluko et al., 2004). The hexameric structure of 11/12S globulin has shown reversible dissociation and association
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Chapter 1 Introduction due to change in pH and ionic strength of the medium. The hexameric structure is maintained when ionic strength is above 0.5 M while hexamer is dissociated into 7S trimers on lowering the ionic strength. When ionic strength is increased, the dissociated 7S trimers can reassemble again (Schwenke et al., 1983).
These proteins show foaming, emulsification , water absorption, fat absorption and protein dispersibility index characteristics (Wu & Muir, 2008). Similarly, the result of in- silico analysis showed that several peptide sequences in cruciferin proteins have biological activities like anti-oxidative, antithrombotic, antibacterial and antifungal (Wanasundara, 2011). Storage proteins have allergenic properties which make them important for clinical practice (Menéndez et al., 1988). Bhatty et al. (1968) reported that the 12S globulin from seeds of Brassica napus was commercially extracted by the use of 10 % weight by volume (w/v) NaCl solution and precipitation of protein was done with a change of pH. Dialysis and Sephadex G-100 gel filtration can be done for further purification of proteins (Bhatty et al., 1968). Schwenke et al. (1981) reported that the precipitation of raw globulin exists during dialysis of 5 % (w/v) NaCl extracted protein solution against water. The 1.7S (Albumin or Napin) and 12S (globulin or Cruciferin) are present in the precipitated protein. The low molecular weight 2S Albumins of Brassica napus were firstly reported by Bhatty et al. (1968) by extraction with 0.01 M buffer of sodium pyrophosphate at pH 7.0 and further chromatographic purification was done by the use of Sephadex G-75 and Sephadex G-100.
1.2.2 Ubiquitins
Ubiquitin is a globular and small protein present in all eukaryotic cells and even prokaryotic cells (Durner & Böger, 1995). The amino acid sequence of this protein is highly conserved from protozoa to vertebrates (Hochstrasser, 2000). Ubiquitin-protein has 76 amino acid residues and has a molecular weight approximately 8.5 kDa (Thrower et al., 2000). Ubiquitin acts as a covalent modifier of proteins in diverse post-translational regulatory mechanisms that are present in all eukaryotic cells (Haglund & Dikic, 2005).
The last four C-terminal residues (Leu-Arg-Gly-Gly) expand from the compact structure and involve in a 'tail' formation, which is essential for its function (Mukhopadhyay & Riezman, 2007). Ubquitin is covalently binding to a substrate protein in the form of signal molecule and due this attachment give the different outcomes.
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Chapter 1 Introduction
Ubiquitin is covalently bind through an isopeptide bond formed between the C-terminal of glycine amino acid and the epsilon amino group of lysine amino acids in the mark proteins (Peng et al., 2003). Additionally, covalent mitigation of proteins with ubiquitin (Ub) has appeared as a powerful method that regulates the stability, function or confine to multiple proteins (Pickart, 2001). There are other types of ubiquitination, such as mono ubiquitination and non-canonical ubiquitination, are involve in different many cellular functions, including DNA damage repair, endocytosis, signal transduction and endosomal sorting (Jurado et al., 2008). Other than these important functions, ubiquitins are also involved in antigen process, apoptosis, Immune response and inflammation ribosome biogenesis (Kurosu et al., 2007). Three enzymes; Ub-activating enzyme (E1), Ub- conjugating enzyme (E2 or UBC) and Ub ligase (E3) play the sequential role in ubiquitination of objective proteins (Love et al., 2005)
The latter is mediating the Ubiquitin is covalently bind through an isopeptide bond between the C-terminal glycine and the epsilon amino group of lysine amino acids in the mark proteins (Peng et al., 2003). One or more ubiquitin molecules are covalently bound to a substrate protein as a signal molecule at one or more lysin conjunction sites. This attachment produced to various outcomes, predominantly depending on the ubiquitination pattern. The widely known end of ubiquitinated proteins is degradation by the 26S proteasome, one particular case being that more than four Ubs make a multi-Ub chain through lysine K-48 amino acids (Thrower et al., 2000).
1.2.3 Napins (2S Albumin)
Napin (2S Albumin) is a low molecular mass protein present in Brassicaceae oilseed and belongs to the prolamin superfamily (Shewry et al., 1995). In Brassicaceae oilseed storage protein, Napin is the second most abundant protein after Cruciferin and constitute about 15 to 45% of SSPs depending on the Brassicaceae species and variety. The Napin precursor is about 21 kDa, but after the seed maturation it has a molecular mass in the range of 12 to 16 kDa and is composed of two polypeptides chains of 4.5 and 10 kDa (Schmidt et al., 2004). In the expression of Napin, multiple genes are involved e.g., NapA, NapB, BngNAP1, gNa and three cDND sequences pN1, pN2 and pNAP1 (later identified as NapA) genes have been identified out of these 10-16 multigenes. These genes are responsible for the expression of Napin in Brassica. napus (Gehrig &
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Chapter 1 Introduction
Biemann, 1995). Napin proteins have basic nature in Brassicaceae plants. It has an isoelectric point (pI) approximately around pH 11 (pI ~ 11) due to the high content of glutamine and asparagine (Lonnerdal et al., 1972). Brassica napus napins are heterodimer of two polypeptides having pH 8.8 and 9.4 as their isoelectric points (Schmidt et al., 2004). These two polypeptides are bound through disulfide linkage (Krzyzaniak et al., 1998).
Venkatesh & Rao (1988) reported the molecular weight of B. juncea Napins in the range of 12 to 13 kDa and is further made up of two polypeptides of approximately 4 and 9 kDa. Napin of S. alba has a molecular mass around 14.6 kDa and has two polypeptide chains i.e., 9.5 and 5 kDa, according to Menendez et al., (1988) and Venkatesh & Rao (1988). In three-dimensional (3D) molecular structure determination of Napin, disulfide bonds play a crucial role. Cysteine residues are the important amino acid in the polypeptide chains and are considered as conserved features of Napin due to their location and number of cysteine residues in the polypeptide chain. There are eight cysteine amino acids (8 Cys motif) that have been pinpoint in different Napin isoforms; six in the long chain and two in the short chain (D'Hondt et al., 1993)
Four disulfide bonds are formed between two chains because of the close proximity of Cysteine amino acid in the polypeptide chain which allows the formation of a solid structure that is difficult to enter by digestive enzymes (Gehrig & Biemann, 1995). Some isoforms of Napin are considered as allergenic like Sin a 1 of S. alba Napin of is soluble in less ionic strength solution or water and as well as in neutral salt solutions (Monsalve & Rodriguez, 1990). Previous work (Bhatty, 1972; Bhatty & Finlayson, 1973; Gillberg & Tornell, 1976; Lonnerdal et al., 1977; Raab & Schwenke, 1984) indicated that the Napins or albumin part can be purified by heat coagulation, and precipitation was done by salting out procedure (using Sodium Chloride, Ammonium Sulfate, etc.) phenomenon and complicated formation with polyionic compounds (alginate, pectinate, carboxymethylcellulose, polyphosphate), ethanol treatment, floculating agents (polyacids, tanning agents), polyamides, polyalcohols and strong acids (trichloroacetic acid, sulfosalicylic acid).
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Chapter 1 Introduction
1.2.4 Thionins
Thionins have been isolated in plants such as barley, wheat and Arabidopsis thaliana (Asano et al., 2013; Florack & Stiekema, 1994; Ozaki et al., 1980). These proteins show antimicrobial activities and have a conserved cysteine-rich region. On the basis of their 3D structure, there are two groups of thionins which are α/β thionins and γ thionins (Pelegrini & Franco, 2005). A C-terminal coil region, two α-helixes and a double-stranded β-sheet are present in the α/β thionins (Pelegrini & Franco, 2005). One α helix and three anti-parallel β-sheets exist in the γ thionins, forming a typical amphipathic α/β two layer sandwich (Pelegrini & Franco, 2005). Antimicrobial proteins of plants can be used against the antibiotic defiance microorganisms in order to develop new drugs (Simoes et al., 2009). Plant-derived antimicrobial compounds can provide clinical value for the therapy of infections caused by several fungi as well (Stein et al., 2005). Specifically, some thionins possess cytotoxic effects whereby they can be applied in the development of new anti-cancerous drugs (Florack & Stiekema, 1994). Transgenic plants having higher levels of Thionins; are more pathogen resistant and reduced the crop losses in agriculture (Pelegrini & Franco, 2005). In short, the antimicrobial activities of Thionins are most promising to develop novel antibiotics and structural characterization of these proteins could give peculiar drug candidates towards human pathogens (Thomma et al., 2003).
1.3 Losses due to pathogen attack
Fungal and Insect attacks can damage cash crops very badly and is a potential ongoing threat for farmers. 25-50% crop losses occur every year from varieties of insects and microbes causing plant diseases. A food is the result of photosynthesis; therefore crop species are primary part in this process and should be saved from pathogens and insects. Foliar fungal diseases are most common in plants like downy mildew, rust and powdery mildew. They are severe airborne disease issues in fruits and cereals plants. Therefore, a wide-range secure system should be needed against microbial phytopathogens to decrease or even abolish crop yield as well as dependence upon chemical pesticides, which expand farmer‘s fixed costs world widely which is especially not comfortable to smallholder farmers in developing countries. As each species can be damaged by the attack of different pathogens. It is necessary to discover plant originated compounds with great
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Chapter 1 Introduction defiance against different pathogens and development of such wide range disease defiance will save time and will be a requirement to develop defiance for particular individual pathogen.
1.4 Aims and Objectives
The purpose of this study was to explore important Brassicaceae genera/species regarding the identification and characterization of their important seed proteins. Different proteins are anticipated to show different features that are distinctive for each protein. The study was planned to obtain highly pure proteins followed by their thorough structural and functional characterization. In particular, discovery of pathogen resistance proteins could make crop resistance by plant transformation against the wide range of fungal and insecticidal pathogens. In compliance to this scenario, the present study was designed targeting the following aims and objectives: Screening of Brassicaceae genera/species for proteomic analysis. Purification and structural characterization of the selected proteins. Functional characterization of the selected proteins targeting their antifungal, anti- cancerous and insecticidal potentials.
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MATERIALS AND METHODS
Chapter 2 Materials a &Methods
2.0 Screening of Brassicaceae species
The following different plants of family Brassicaceae were screened for the identification and characterization of important proteins as shown in Table 1. The proteins were isolated in 0.1 M Tris buffer of pH 7.0 from seeds of enlisted plant species.
Table 1. List of different Brassicaceae species used for screening of important proteins.
Sr. No. Scientific name Organ used 1 Brassica campestris Seeds 2 Brassica juncea Seeds 3 Brassica napus Seeds 4 Brassica nigra Seeds 5 Brassica oleracea Seeds 6 Brassica rapa Seeds 7 Eruca sativa Seeds 8 Iberis amara Seeds 9 Iberis umbellata Seeds 10 Iberis gibraltarica Seeds 11 Lepidium capitatum Seeds 12 Lepidium sativum Seeds 13 Lobularia maritima Seeds 14 Nasturtium microphyllum Seeds 15 Raphanus raphanistrum Seeds 16 Sinapis alba Seeds 17 Sisymbrium irio Seeds 18 Sisymbrium loeselii Seeds
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Chapter 2 Materials a &Methods
2.1 Preparation of seed crude extracts
Seeds of plants were thoroughly washed with distilled water to remove dust or mud. Every individual material was weighed equally to 10 grams using an electronic balance (AX200, Shimadzu) and was powdered by using a blender or pestle and mortar after freezing in liquid nitrogen or directly in a grinder. Extraction procedure was carried out in two steps. In the first step, the water insoluble components were removed by treating fine powder with 80 % ethanol. Ethanol was removed by filtration and the residue was air-dried for further extraction. The protein extractions were performed at temperature 25 °C and then each extraction was done at four different pH values (0.1 M Citrate buffer, pH 3.0; 0.1 M Acetate buffer, pH 5.0; 0.1 M Phosphate buffer, pH 7.0 and 0.1 M Carbonate buffer, pH 10.0) while 1 mM PMSF was added as protease inhibitor. All buffering solutions were prepared as per standard protocols. Corresponding pH values and molar strengths of different buffers have been mentioned where needed. The extracts were centrifuged at 16,000 × g for 25 minutes. The pellet was discarded and the collected supernatant was further filtered to remove the any particulate material or debris present in the supernatant.
2.2 Protein quantification
2.2.1 Bradford reagent
The protein content of the samples were calculated by Bradford technique (Bradford, 1976) against Bovine Serum Albumin as standard and the change in color was measured Spectrophotometrically (BMS Spectrophotometer, SN 204573) at wavelength (595 nm).
2.2.2 Nanodrop 2000c
The proteins were also quantified by applying a Nanodrop 2000c instrument (Thermo Scientific, peqLab, Germany). 2 μl of the corresponding buffer was used as blank. Absorbance was converted to corresponding quantifications by the help of Lambert and Beer equation as given below:
A280 = ɛ * b * c
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Chapter 2 Materials a &Methods
The theoretical molar extinction coefficient ε [M-1 cm-1] was determined by ProtParam (Gasteiger et al., 2005).
2.3 Gel electrophoresis
2.3.1 One dimensional SDS-PAGE
To visualize the protein extraction as well as the purity, one dimensional SDS- PAGE was performed for each sample. The common procedure (Laemmli, 1970) was acquired for the preparation of the 12 % and 15 % gels (E-VS10-SYS, omniPAGE mini-System) to visualize the protein samples. Non-reduced and reduced samples (β- mercaptoethanol) were heated at 95 °C for 5 minutes in the heating block to denature the samples. The gel was stained with Coomassie dye (CBBR-250) (Sambrook et al.,1989) for visualizing the protein bands.
2.3.2 Two dimensional (IEF/SDS-PAGE)
For two dimensional (IEF/SDS-PAGE) electrophoresis, protein concentration was calculated using Pierce™ BCA protein estimation kit. After cleanup, approx. 80 휇g of protein material was dissolved in rehydration buffer containing 8 M Urea, 0.5 % ampholyte solution, 0.2 % DTT, 0.5 % CHAPS and rehydrate the IPG strip 3–10 NL overnight. IEF was accomplished applying Multiphor II (GE Healthcare) system at 20 ºC applying 7000 V/h. IPG Strips were equilibrated for 30 minutes each with DTT and 2- Iodoacetamide, respectively. After equilibration, strip was applied on 12 % polyacrylamide gel to run second dimension of SDS-PAGE at a constant voltage of 65 volts using mini protein electrophoresis system (Bio-Rad). Gels were stained overnight using Coomassie dye.
2.4 Protein identifications
2.4.1 Electroblotting
Polyvinylidene diflouride (PVDF) membrane (Standard Millipore Immobilon 0.45 micrometer membrane) blot was prepared according to the standard protocol (Towbin et al., 1979). The transfer of protein bands was performed at 200 mA for 4h.
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Chapter 2 Materials a &Methods
2.4.2 N-Terminal amino acid sequencing
A N-terminal amino acid sequence was obtained applying the peptide and protein sequencing system (Applied Biosystems Edman sequencer 476, Germany) using Edman degradation methodology (Hewick et al., 1981). A Search was performed by applying Protein BLAST in UniProtKB protein database (http://www.uniprot.org/blast/) for related alignments sequences and homologous sequences.
2.4.3 Mass-spectrometry of proteins
2.4.3.1 MALDI-TOF/TOF Mass-spectrometry
Gel pieces of 2 mm3 thickness were excised and transferred into sterile micro axis tubes. Trypsin was used to digest the protein and peptides were taken onto a MALDI plate which was blended for an equivalent volume of α-cyano-4-hydroxylcinnamic acid matrix (10 mg/1000 µl) in 0.1 % TFA and 50 % CAN. Utilizing a MALDI-TOF/TOF 5800 mass spectrometer (ABSciex, CA) working in reflectron mode over a window of 700 m/z to 4,000 m/z, Peptide Mass Spectrum was obtained through the Protein Pilot software 4.0.
2.4.3.2 LC–MS/MS Mass spectrometry
LC–MS/MS calculations were taken by pouring the samples on a nano liquid chromatographic system (Dionex Ultimate 3000) coupled through the electrospray- ionization (ESI) to an orbit-rap mass spectrometer (Orbitrap Fusion, Thermo Scientific, Bremen, Germany). The samples were filled (5 μl/ 60 second) on a catching column
(Acclaim PepMap μ-precolumn, C18; buffer A: 0.1% formic acid in H2O; buffer B: 0.1 % formic acid in acetonitrile) with 2 % buffer B, cleaned for 5 min with 2 % buffer B (5 μl/min) and the peptides were eluted (200 nl/min) onto the dissociation column (Acclaim PepMap 100, C18, 75 μm×250 mm, gradient: 2–30 % B in 30 min). LC–MS/MS examine was executed in data dependent acquisition mode (DDA). Spectra of MS/MS were documented in an ion trap instrument. LC–MS raw data were operated with Proteome Discoverer 2.0 (Thermo Scientific, Bremen, Germany). MS/MS spectra were explored with Sequest HT against the Arabidopsis in UniProtKB protein database (http://www.uniprot.org/blast/) for identification.
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Chapter 2 Materials a &Methods
2.5 Protein purification
Identified seed proteins of different genera were further purified to homogeneity by different strategies. Different saturation constants of ammonium sulfate precipitations were performed according to Duong & Gabelli, 2014 as a first step of purification. SDS- PAGE was run to analyze the partial protein purification and their respective molecular weight determination. The suitable fractions were dialyzed using the dialysis membrane of appropriate MWCO 3.5 kDa pore size to remove the ammonium sulfate salt from the protein samples.
2.5.1 Fast Protein Liquid Chromatography (FPLC)
After dialysis, the protein samples were applied to an ÄKTA FPLC purification system (ÄKTA Purifier P-901; GE Healthcare Life Sciences, UK) machine containing cationic column Mono S 5/50 GL (GE Healthcare) which was already equilibrated with corresponding buffer A. The column was cleaned with many bed volumes of the equilibrated buffer and then with corresponding buffer B (buffer A with 150 mM NaCl) to elute the desired proteins. The peaks were examined by SDS-PAGE and purified fractions were pooled to check the final purity. The pooled sample were loaded on a Gel filtration column Hi-Load 16/60 Superdex 200; Hi-Load 16/60 Superdex 75 (GE healthcare) to remove minor impurities and to obtain more than 95 % purity. The bound fractions eluted from the column were dialyzed against buffer A and then was subjected to SDS-PAGE for purity analysis and purified fractions were pooled together.
2.6 Structural Characterization
2.6.1 Circular Dichroism (CD) spectroscopy
CD spectroscopy is an approach applied to determine the secondary structure of peptides, proteins and polypeptides in solution, as well as investigating the stability of proteins by changing buffer, the melting temperature and folding changes after addition of additives. The CD-spectroscopic experiments were performed in 1 mm quartz cuvettes with a total volume of 0.1 ml of 2-10 µM protein solution on a Jasco J-815 instrument (Jobin Yvon, Longjumeau, France). 15 measurements were applied for far-UV experiments. The wavelength range of 260-190 nm was carried out with an interval of 0.1
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Chapter 2 Materials a &Methods nm, which were combined by arithmetic averaging. The buffer was measured by the same procedure and subtracted from the protein signal. The protein was centrifuged at 4 °C for 30 min at 15,000 rpm prior to measurement. For the thermal stability of the protein, the melting curve with a heating up to 1 °C/min was applied, the changes in CD signal at 208 nm was detected. The elasticity θ is defined as the difference in absorbance of clockwise and counter clockwise circularly polarized light. The percentage of the secondary structure of the protein was determined by using Spectra manager TM software (Jasco).
2.6.2 Dynamic Light Scattering (DLS) measurements
Purified protein was analyzed by Dynamic Light Scattering (DLS) using the spectroLight 300 (Xtal Concept) confirming the mono dispersity of the protein solution and approximating the size of the protein. The hydrodynamic radius of native protein was calculated as well. A 20 µl sample of pure transparent protein was used to take a series of measurements with a sampling time of 45 s and a laser light of 659 nm was used. A fixed angle of 90o was used to collect the scattered light. The autocorrelation function to particle size distribution was determined by program CONTIN (Provencher, 1982). DLS measurements were performed minimum twenty times and the values were calculated with standard error.
2.6.3 Small Angle X-ray Scattering (SAXS)
Biological SAXS has become a popular technique to characterize shape, overall structure and molecular weight of nucleic acids, macromolecular complexes ,proteins and peptides in solution (Hura et al., 2013; Svergun et al., 2013). The elastic scattering of x- rays with wavelength 0.1-0.2 nm is recorded at low angles (0.1 - 10°) and analyzed by the sample. Partially dissociating complexes or oligomers, the soluble aggregates and contaminants should be removed to obtain good SAXS result. For 3D structural analysis, monodisperse and homogenous solution is required (Jacques et al., 2012). The monodisperse solution obtained by the help of Linear Guinier plots. The sample is filtered and checked by DLS prior to SAXS analysis.
Monodisperse solutions of protein at three different concentrations in corresponding buffers, were poised at EMBL beamline P12 at storage ring PETRA III (DESY, Hamburg, Germany) in 2016 (Blanchet et al., 2015). At a sample-detector has
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Chapter 2 Materials a &Methods distance of 3.1 m and λ = 0.124 nm wavelength, scattering data were poised applying a 2D photon-counting Pilatus 2M pixel detector (Dectris) with the momentum transfer and it has a range from 0.03 nm−1 < s < 4.8 nm−1 (s = 4π sinθ/λ, where 2θ is the scattering angle). To monitor for radiation damage, 20 successive 45 MS X-ray submission of protein were differentiated and no changes in the intensity pattern were observed. Data were normalized by using the beam intensity and averaged symmetry. After subtraction of the buffer scattering, the different curves were scaled for concentration of proteins. The radius of gyration Rg along with the particle pair-distance distribution function p(r) which further provides the maximum dimension Dmax were calculated by the self-operating SAXS data examine pipeline SASFLOW (Petoukhov et al., 2012). Low-resolution ab initio shapes of protein were generated based on the composite scattering curves applying the program DAMMIF (Franke & Svergun, 2009). It uses a group of densely filled beads representing the particle shape and involves motivated annealing to build a model that fits the experimental scattering intensity data. Ten DAMMIF was complete in order to verify solution stability and well-superimposable models as judged based on their normalized spatial discrepancy (NSD) values. CRYSOL was (Svergun et al., 1995) used to make superposition or comparisons between scattering data and known high-resolution structures. CRYSOL is aware of the hydration shell and calculates a spherically averaged scattering pattern using multipole expansion of the scattering amplitudes. Varying the background, hydration shell contrast and the relative amount of displaced solvent, the χ - value is minimized. This value comprises a quality measure of the deviation of the fitted function and PRIMUS (Konarev et al., 2003) were applied both for rigid-body modeling.
2.7 Crystallization of proteins
2.7.1 Pre-Crystallization test
Protein concentration for crystallization screening was determined by using the Pre- Crystallization Test (PCT, Hampton Research). The amorphous precipitate was formed as result of highly concentrated protein samples, while diluted protein samples had produced transparent drops. Clear drop production and the amorphous precipitate were avoided by changing the protein concentration accordingly in order to get the successful homogenously distributed light granular precipitates of target protein.
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Chapter 2 Materials a &Methods
2.7.2 Robotic screening of crystallization conditions
The suitable protein concentrations for crystallization were optimized by concentrating it in crosspending buffer using suitable (Amicon Ultra-15, Millipore) concentrating tubes. Crystallization screening was accomplished using a Honey bee 961 dispensing robot (Zinsser Analytical GmBH, Frankfurt, Germany) appealing the sitting- drop vapour-diffusion method at 293 K in 96-well plates (NeXtal QIA1 µplates, Qiagen) as shown in Fig. 1. In initial crystallization screening, JCSG, Classic, Cryos, ComPAS, Ammonium Sulphate (Qiagen, Germany), Pact, Morpheus and Stura suites (Molecular Dimensions, UK) were screened. Initial crystals were got from different conditions and then these conditions were optimized by vapor diffusion method.
Fig. 1. Pipetting robot for putting different crystallization conditions (Zinsser Analytical GmBH, Frankfurt, Germany).
2.7.3 Optimization of initial crystallization conditions
Hanging Drop Vapor Diffusion method (Patrick, 2007) was used to optimize the initial crystallization conditions to improve initially obtained crystals. Conditions were
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Chapter 2 Materials a &Methods optimized in 24-well Linbro plates (Hampton Research, USA). A drop has 2 µl proteins and 2 µl reservoirs were equilibrated against 1 ml reservoir.
2.7.4 Confirmation of protein crystals
Protein crystals were evaluated by using the CystalScore (Diversified Scientific Inc., USA) and SONICC (Benchtop, Formulatrix. Inc, USA) to distinguish from salt crystals
2.7.5 Data collection and processing
Diffraction data from crystal was collected by exposing it at the Synchrotron P11 and P13 (Beam lines at Petra III) Deutsches Elektronen-Synchrotron (DESY), Hamburg with PILATUS 6 M detector to characterize the crystals. The data was collected with 0.6 oscillation range per frame. A crystal was fished by help of nylon loop. It was flash- cooled in cold nitrogen gas stream at 100 K. Diffraction data from single crystals was analyzed by several computer programs. Diffraction data was processed with XDS (Kabsch, 2010) iMOSFLM or HKL2000/ Denzo (Otwinowski & Minor, 1997).
2.8 In-Silico analysis
The obtained peptide fragments were submitted to Quick 2D (Alva et al., 2016) server for the prediction of secondary structural elements. Each individual peptide identified by LC–MS/MS or MALDI–TOF/TOF was searched using BLAST www.uniprot.org in the UniProtKB/Swiss-Prot database for their recognition. A search was also executed by using a BLAST to multiple sequence alignment for amino acid sequences in the UniProtKB/Swiss-Prot database. Similarly, for the calculation of predicted protein models, a search was performed in Protein Data Bank (PDB) online server (www.rscb.org). Non-redundant sequences from different species were subsequently aligned using ClustalW2 employing the default parameters (Thompson et al., 1994). The crystal structure of an ubiquitin from Saccharomyces cerevisiae (Uba1, PDB-ID: 4NNJ_B) was found as the best template by providing 90 % sequence identity, 56 % similarity and an expected value of 1e-24. The aligned sequences of Siu and Uba1 were subjected to model building in Swiss-Model server (Arnold et al., 2006; Biasini et al., 2014; Guex & Peitsch, 1997). Models were constructed based on the target-template
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Chapter 2 Materials a &Methods alignment using ProMod3 server (Benkert et al., 2011). Coordinates which were preserved between the target and the template were duplicated from the template to the model. Remodeled was made by the process of insertions and deletions with the help of using a fragment library and after this side chains were reconstructed. Finally, with the help of force field, the geometry and shape of the resulting model was regularized. The quality of the model was assessed by drawing Ramachandran plot using Procheck (http://services.mbi.ucla.edu/PROCHECK/) (Laskowski et al., 2001). Figures containing structural details were rendered by using Chimera software (Pettersen et al., 2004).
2.9 Bioactivity assays
2.9.1 Cell cytotoxicity assay
MTT assay kit (Millipore, USA) was used for rapid and perceptive quantification of cell proliferation and viability. Briefly, 100 µL (1×105) of Huh-7 cells were cultured in a 96 wells plate using the Dulbecco‘s modified Eagle medium (DMEM) was used as accessory with 10 % fetal bovine serum and 100 IU/ml penicillin and 0.1 mg/1000 μl streptomycin at 37 °C in a carbon dioxide (CO2) incubator for 24 hours. Different dilutions of precursor Eruca sativa Thionin (pEsT) protein was added separately and these plates were incubated at 37 °C in a carbon dioxide (CO2) incubator for another 24 hours and three replications were analyzed for each dilution of each protein. After 24 hours, the medium was removed and100 µl freshly prepared medium was added along with 10 µl (3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide) solution in 5 mg/ml phosphate buffered saline (PBS) as per manufacturer‘s instructions. The plate was again incubated in a carbon dioxide (CO2) incubator at 37 ºC for 4 h and after this 0.1 ml Dimethyl sulfoxide (DMSO) was put in to disappear the formazan crystals in the wells. The MTT formazan product was detected by measuring the optical density with a multi-channel plate reading photometer at a test 570 nm wavelength and a reference 620 nm wavelength. The Cell feasibility was attained by means of the following formula (Rehman et al., 2011).
( ) ( ) [ ] . ( ) ( )
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Chapter 2 Materials a &Methods
2.9.2 Antifungal assay
Antifungal activity was executed by Disc Diffusion method (Boyle et al., 1973) against Fusarium solani and Fusarium oxysporum while 96-well microtiter plate (Martin & Wiederrecht, 1996) against Fusarium graminearum.
2.9.2.1 Disc Diffusion method
This method was executed on two fungal species Fusarium solani and Fusarium oxysporum. A triplicate of 10 ml potato dextrose agar was prepared in Petri dishes. After mycelial colony had grown, sterile blank paper discs (0.625 cm in diameter) were kept at equidistance from the mycelial colony growth. Optimized concentrations of the purified proteins (1 μg/μl) were applied on the discs. These plates were incubated at 23 °C for 72 h till mycelial growth. The paper discs soaked with buffer were the negative control while the fungicide (TOPSIN® 4.5FL) disc was set as positive control. There was appearance of inhibition around the discs carrying pEsT or BnN proteins.
2.9.2.2 96-well microtiter plate
The 96-well microtiter plate assay is a sensitive and fast method for large scale measurement of the inhibitory effect of antifungal substances in vitro. With the fluorometric analysis we are able to detect growing hyphae which are constitutively expressing GFP. The fluorescence signal intensity correlates here with the growth rate of hyphae. To determine the antifungal substances‘ inhibitory impact we measured the hyphal growth over a time period of 120 h. A 96-well plate assay was used to measure the impact of the protein Thionin on the germination and growth of the fungus Fusarium graminearum. A well was containing 100 µl Minimal Medium (MM), 4 µl conidia, three concentrations of the pEsT protein and BSA (30, 50 and 100 µg in corresponding wells) in addition to buffer and column-flow (CF) as control.
In the First step, the conidia were thawed slowly at room temperature. During the thawing step, minimal medium and the other components were added to their respective wells. After loading the plate, it was sealed off with a special polyester foil. Afterwards, the first measurement of 0 h was recorded by using the Mithras² LB 943 Monochromator Multimode Reader. The excitation occurred with light of wavelength of 460 ± 10 nm and
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Chapter 2 Materials a &Methods the detection of the emission within a range of 520 ± 10 nm. After three measurements the plate was placed in the shaker (142 rpm) at 28 °C and incubated in the dark. The plate was measured every 24 h for 120 h.
2.9.3 Entomotoxicity assay
Entomotoxicity assays of pEsT or BnN were performed in the eco-toxicology laboratory, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan. Thionin/Napin toxicity was determined for Tribolium castaneum insect. A population of T. castaneum was collected from a local flour mill and the culture was grown on wheat flour with 5 % brewer yeast (Khalequzzaman et al., 1994). To get an equal age insect community, the culture medium was reared at 60-90 °C for about one hour. A jar was filled with 500 g flour and 50 red flour beetles were added in it for oviposition. Beetles were removed from the jar after three days with the help of sieves and then were kept in the separate set of sterilized jars. These jars were packed with 200 g flour for preservation of the culture. Eggs were hatched to get adult beetles of a homogenous population. The culture was preserved under optimum laboratory conditions at 30 °C with a relative humidity of 70 %.
For the bioassay, three different serial dilutions of the pEsT protein were added in 0.1 M phosphate buffer of pH 7.0. However, for the insecticidal activity of BnN, 20 mM acetate buffer; pH 5.5 was used. Concentrations of the proteins were 3 mg/ml, 2 mg/ml and 1 mg/ml respectively. 450 g of wheat flour was kept at 4 °C to keep away from any infestation. Homogenous dough was obtained by adding the each protein concentration in 100 ml buffer and after this it was mixed with 150 g flour. Hard pan was prepared by drying the dough in darkness. Fine powder was obtained by grinding the dried dough in electric grinder. Five replications were used for all three protein concentrations and negative control was combined with buffer only. Each replication was executed in an individual glass jar. Five male and five female individuals of Tribolium castaneum were free in each jar. Adults Tribolium castaneum were removed after ten days and interval data of larvae, male and female pupae involving adults was recorded weekly. Data were compared with the negative control to evaluate the effect of protein towards T. castaneum.
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2.9.3.1 Statistical analysis
Antifungal activity of pEsT was performed in 96-well microtiter plate. The mean value of all eight technical replicates from all three measurements (in total twenty-four readings) was calculated. The standard deviation is based on the mean value of all twenty-four replicates. The entomotoxin protein bioassay data was analyzed in one way ANOVA through the stat software ―Statistics 8.1‖ and means were separated by Tukey-HSD test with a significance level of 0.05. The standard error of each treatment was computed through Microsoft Excel.
2.10 Software and Bioinformatics tools
2.10.1 ProtParam
ProtParam is an appliance used to calculate the many chemical and physical characteristics of proteins and amino acid composition molecular weight (MW), atomic composition, molar extinction coefficient, instability index, grand average and aliphatic index of given protein sequence (https://web.expasy.org/protparam).
2.10.2 Multiple sequence alignment
Clustal Omega and Clustal W2 is a multiple sequence alignment site used for the alignments between three or more nucleotide or protein polypeptide sequences (https://www.ebi.ac.uk/Tools/msa/clustalw2/, http://www.clustal.org/omega).
2.10.3 BOXSHADE server
BOXSHADE is used for beautiful printing of multiple alignment outputs. A multiple alignment prepared on Clustal W2 server was used as input to program BOXSHADE (https://embnet.vital-it.ch/software/BOX_form.html).
2.10.4 ATSAS
For small-angle scattering data analysis, an ATSAS suite was used to determine the shape, molecular weight (MW) and overall structure of biological macromolecule (Petoukhov et al., 2012).
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Chapter 2 Materials a &Methods
2.10.5 PDBsum
PDBsum is a database by giving a graphical summarizes information on the macromolecular structure. This information includes protein secondary structure, images of the structure, detailed structural examination, and annotated plots of each protein chains. (https://www.ebi.ac.uk/thornton-srv/databases/cgibin/pdbsum)
2.10.6 PROCHECK
PROCHECK is a suite of programs to check the stereo chemical properties of protein structures. (https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/)
2.10.7 SWISS-MODEL server
SWISS-MODEL is a web-server to structural bioinformatics and homology modeling for the calculation of molecular structures (Arnold et al., 2006; Biasini et al., 2014; Guex & Peitsch, 1997) (https://swissmodel.expasy.org/)
2.10.8 Quick 2D server
The quick 2D server is the MPI bioinformatics is Toolkit that act as a platform for protein sequence and structure examination (Alva et al., 2016).
2.10.9 PyMOL
PyMOL is one of visualization tools in structural biology. PYMOL is used to produce high-quality 3D images of biological macromolecules, such as nucleic acids, proteins and small molecules (DeLano, 2002).
2.10.10 Chimera
Chimera is used for analysis of molecular structures, interactive visualization- related data, supramolecular assemblies, including density maps, docking results, sequence alignments and trajectories (Pettersen et al., 2004).
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RESULTS
Chapter 3 Results
Seed storage proteins are important in providing nitrogen and sulfur during seed germination. Cruciferins (12S globulins) are abundantly occurring storage proteins and belong to Cupin superfamily of proteins. Spectroscopic techniques like Circular Dichroism (CD), Dynamic Light Scattering (DLS) and Small Angle X-ray Scattering (SAXS) indicated interesting results especially about the oligomerization of Cruciferins. Similarly, many other smaller molecular weight proteins like Napins, Thionins and Ubiquitins are also present in seeds of family Brassicaceae. All above mentioned proteins were analyzed thoroughly regarding their biochemical and functional characterization. Low molecular weight proteins showed good inhibitory effects against different fungal pathogens. Additionally, Thionins exhibited good cytotoxic activity against hepatic cancerous line Huh-7 as well as promising insecticidal efficacy.
3.1 Initial screening
Initially 18 different species from Brassicaceae family were screened in search of important seed proteins. Screening was based on the physical appearance of the crude extract, protein banding pattern on SDS-PAGE, thorough literature review and successful results of N-terminal amino acid sequencing or mass spectrometry data. Fig. 2 is showing the protein banding pattern of seed crude extracts of eighteen plants ran under non- reduced conditions of the SDS-PAGE.
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Fig. 2. SDS-PAGE showing crude protein profiles of eighteen different plant species. Crude extracts were prepared in Tris buffer (100 mM, pH 7.5) and were loaded on SDS-PAGE under non-reduced condition.
Lane 1: (Brassica nigra), 2 (Eruca sativa), 3 (Brassica napus), Ml (Protein ledder), 5 (Sisymbrium irio), 6 (Lepidium sativum), 7 (Brassica juncea), 8 (Brassica rapa), 9 (Sinapis alba), 10 (Sisymbrium loeselii), 11
(Brassica campestris), 12 (Lepidium capitatum), 13 (Raphanus raphanistrum), M2 (Protein ledder), 15 (Nasturtium microphyllum), 16 (Iberis amara), 17 (Lobularia maritima ), 18 (Brassica oleracea), 19 (Iberis umbellata), M3 (Protein ledder), 21 (Iberis gibraltarica).
From the result of initial screening, three species were selected for this research work as enlisted in Table 2.
Table 2. List of selected plants and seed proteins that have been identified and characterized among enlisted genera of Brassicaceae family.
Local Seed Storage Sr. No. Botanical Name Other Proteins Name Proteins Probable Cruciferin & 1 Brassica nigra Black Rai ------Probable Napin
2 Eruca sativa Tara Mera Probable Cruciferin Probable Thionin
3 Sisymbrium irio Khob kalana ------Probale Ubiquitin
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Chapter 3 Results
Fig. 3. Seeds of three selected Brassicaceae species. (A) Brassica nigra (B) Eruca sativa (C) Sisymbrium irio.
3.2 Identification and characterization of 12S globulins
12S globulins also known as Cruciferins were identified and characterized from Brassica nigra and Eruca sativa seeds.
3.2.1 Extraction and identification of Cruciferin from B. nigra
A primary sequence of 17 amino acids (GLEETICSMRSNEXEDD) from N- terminus was obtained through Edman degradation. Analysis of this sequence via protein BLAST (UniProtKB) further confirmed the protein as 12S globulin and showed a very high identity with other known Cruciferins of the Brassicaceae family as shown in (Table 3). The sequence showed 82 % amino acid homology to that of 12S CRC Cruciferin from Arabidopsis thaliana (Hou et al., 2005). Further homology comparison suggested a high similarity to already known seed storage Cruciferins of the family Brassicaceae. It also showed high homologies with already reported Cruciferins from Raphanus sativus (Depigny et al., 1992), Brassica napus (Rödin et al., 1992) and to 11S globulins from Sinapsis alba (Palomares et al., 2005). Occurrence of Cruciferin in seeds of many
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Chapter 3 Results different species is a good indication of their wide spectrum of occurrence among members of family Brassicaceae.
Table 3. N-terminal sequence comparison showed high homology (82 %) with A. thaliana Cruciferin and similarly with other 11S globulins. Interestingly three different amino acids (highlighted in red) Serine, Asparagine and Glutamic acid were observed among the sequence of B. nigra Cruciferin.
Species Cruciferin Sequence Homology (%) B. nigra Cruciferin GLEETICSMRSNEXEDD In this Thesis A. thaliana 12S CRC GLEETICSMRSHENIDD 82 R. sativus PGCRURSE5 GLEETICSMRTHENIDD 76 B. napus CRU1 GLEETICSMRTHENIDD 75 S. alba 11S Globulins GLEETICSMRTHENIDD 65
3.2.1.1 Purification of Cruciferin
The 12S Cruciferin with its polypeptide chains and the corresponding isoforms were analyzed by one and two-dimensional electrophoresis techniques respectively. Cruciferin was precipitated from the crude extract of defatted powder by ammonium sulfate (30 % saturation) precipitation. Excessive salt was removed by extensive dialysis and partially pure protein was subjected to gel filtration chromatography with PBS buffer pH 6.5 (Fig. 4A) in order to remove further impurities. Ultimately, an optimized combination of ammonium sulfate precipitation along with chromatographic steps provided a 95 % pure Cruciferin protein (Fig. 4B) from seeds of B. nigra. One dimensional SDS-PAGE performed under both reduced and non-reduced conditions helps to further characterize the protein. Under non-reduced condition, it showed one band corresponding to high molecular weight protein of approximately 190 kDa (lanes 1 & 2) which seem to be the trimeric forms of a 50-55 kDa monomer as shown in lane 3 of (Fig. 4B). The corresponding α and β polypeptides were further confirmed by applying the Cruciferin under reduced conditions. Addition of β-mercaptoethanol reduced the 50 kDa monomer releasing acidic and basic chains which are revealing the typical composition of this protein. This separated monomer in two smaller subunit ranges of approximately 28- 32 (α) and 17-20 (β) kDa bands as shown in lanes 4 and 5 of (Fig. 4B).
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Chapter 3 Results
Fig. 4. (A) Gel filtration chromatogram of the pure Cruciferin. (B) SDS-PAGE is showing the different forms of the 50 kDa monomer (L3) e.g., L1 and L2 are indicating the trimeric form while L4 and L5 are showing reduced acidic (α) and basic (β) polypeptides.
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Chapter 3 Results
3.2.1.2 2D Electrophoresis (IEF/SDS-PAGE) of Cruciferin
2D gel electrophoresis proved a useful technique towards identification of hetero- oligomeric subunit pairs and towards establishing relationship with their constituent polypeptide chains in seed proteins. The presence of a mother band with molecular weight of approximately 190 kDa proposed the presence of multiple isoforms hiding under the bands of 30 (α polypeptides) and 20 (β polypeptides) kDa ranges. To further analyze these different isoforms, two-dimensional electrophoresis (IEF/SDS-PAGE) was performed in order to separate these isoforms based on the differences between their isoelectric points. As shown in (Fig. 5), α-polypeptide chains are separated in a molecular weight range between 28 to 32 kDa with pI ranges between 5.7 to 8.0 (red box). Similarly, the β- polypeptide is separated in a molecular weight range between 17 to 20 kDa with a pI range of 5.5 to 8.5 (yellow box).
Fig. 5. Two-dimensional electrophoretic separation of Cruciferin subunits showing the α (red box) and β- polypeptides (yellow box) into their respective isoforms.
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Chapter 3 Results
3.2.1.3 Secondary structure determination of Cruciferin
Circular Dichroism (CD) data suggested the presence of both α-helical and β- sheeted secondary structu6re of the 190 kDa native Cruciferin, showing a broad negative band which is typical for α and β type protein (Manavalan & Johnson, 1983) (Fig. 6A). The secondary structure content was calculated to approximately 30 % α-helical structure, 15 % turns and loops while major part (55 %) is comprised of β-sheets. A comparative topology diagram of B. napus 11S globulins (PDB ID: 3KGL) illustrating the dominating anti-parallel β-strands is also shown in Fig. 6B. The molecular structure of 11S seed globulin from B. napus has been determined and is deposited in Protein Data Bank via code 3KGL. The pro-11S globulin has been expressed in E. coli and was then crystallized to solve the structure through x-ray crystallography. A brief analysis of the structure is showing the dominance of anti-parallel β-sheeted secondary structure over the helices which are just a minor part of the molecule as shown in (Fig. 6B). Similar properties have been observed in the CD spectra of B. nigra Cruciferin as the characteristic negative band at 222 nm from α-helices was not observed, in accordance with a lower content of α- helical structure of Cruciferin (Fig. 6A).
Fig. 6. (A) Circular Dichroism (CD) spectroscopy showing α-helical and dominating β-sheeted conformation. (B) Topology diagram of B. napus 11S globulins (PDB ID: 3KGL) illustrating the dominating anti-parallel β-strands.
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3.2.1.4 Dynamic Light Scattering analysis
Initially, the Cruciferin was extracted and purified in 50 mM phosphate buffer (pH 6.5), however, DLS showed a polydisperse signal as shown in (Fig. 7A) indicating the presence of some high molecular weight oligomers at pH 6.5. Therefore, different buffers were tested through dialysis and ultimately the purified Cruciferin solution was adjusted to a concentration of 12 mg/ml in 20 mM Tris-HCl, pH 7.5. The solution was centrifuged, filtered using micro-spin filter tubes and subjected again to DLS measurements. A single prominent signal corresponds to a monodisperse protein solution (12 mg/ml) with RH of 5.8 ± 0.1 nm as shown in (Fig. 7B and 7C) along with the effect of the buffer on the removal of larger oligomers. A combined effect of pH and salt has been observed to influence the oligomerization of Cruciferins along with other physical factors. Such physical factors are influencing the intra and inter-chain hydrogen bonding of the surface occupied aliphatic side chains. A low ionic strength and pH near to physiological value favors the development of trimer development.
Fig. 7. (A) Dynamic light scattering (DLS) measurement showing polydisperse pure Cruciferin solution in phosphate buffer (pH 6.5). (B) A monodisperse solution dissolved in Tris buffer (pH 7.5). (C) Histogram showing the hydrodynamic radius RH (5.8 ± 0.1 nm) confirming the trimeric status.
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3.2.1.5 Diffraction data analysis
Flower like crystals (approximately 0.1 ×0.09×0.06 mm) appeared after nine months in condition H11 of Morpheus (Molecular Dimensions, UK) containing 10 % PEG 4,000, 20 % (v/v) glycerol and 0.1 M trizma base (pH 8.5) and 0.02 M of each amino acid compounds as shown in (Fig. 8). Unfortunately these crystals diffract up to maximum 8 Å resolutions, which was unsuitable for structure determination and therefore, the data collection was stopped after processing of few images.
Fig. 8. Crystals of purified 12S globulin/Cruciferin of Brassica nigra. The largest dimension of crystal is approximately 100 µm. Scale bar, 0.05 mm.
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3.2.1.6 Analysis of SAXS data
The structural details of these storage proteins are of much interest to understand patterns of their molecular oligomerization and packaging inside the protein vacuoles as well as their functions at the molecular level. The trimeric state of Cruciferin in 20 mM
Tris-HCl, pH 7.5 was analyzed using SAXS (Fig. 9 and 10). The radius of gyration Rg and the maximum dimension Dmax obtained from the collected data were 4.24 ± 0.25 nm and 14.0 ± 0.4 nm, respectively (Fig. 9A and 9B); these values suggest a folded but extended structure. Based on the shape of the Kratky plot curve the protein is rather rigid and compactly folded (Fig. 9B).The scattering amplitude of Cruciferin processed by PRIMUSQT (Petoukhov et al., 2012).was compared to an 11S globulin from B. napus (PDB ID: 3KGL) (Tandang et al., 2010) by the program CRYSOL (Svergun et al., 1995) in Fig. 10. The calculated minimized χ2-value of 1.337 for the comparison of the scattering pattern of Cruciferin (blue) and 11S globulin from B. napus (red) over a wide range of angles confirmed high structural similarity. A comparable molecular weight is suggested by the forward scattering intensity. Even the intensities at moderately higher scattering angles (s > 0.8 nm-1) are matching well pointing at the conservation of some structural detail. A conservation of the trimerization and a globular slightly oblate shape are strongly indicated as further deduced from the p(r) function (Fig. 9C). In addition to these SAXS parameters, all other important values have been summarized in (Table 4).
A tentative ab initio model of Cruciferin (Fig. 10, grey spheres) displays an extended ―loop structure‖ sticking out of the nearly globular surface in agreement with the p(r) function. This feature may correspond to an extended terminus or loop of Cruciferin that possesses some flexibility in solution. Besides, calculated ab initio models are structurally highly similar compared to each other with an NSD value of 1.029 ± 0.112. The displayed ab initio dummy model with P3 symmetry was superimposed with the 11S globulins from B. napus (PDB ID: 3KGL) as prepared by PyMOL (Delano et al., 2002) (Fig. 10). Manual superimposition well confirms the conclusion that the structures are widely similar, including the molecular weight comparison based on the volume of the ab initio model. The shape factor, RG divided by RH as determined by dynamic light scattering, has a value of 0.71 which is indicative for a nearly globular particle in agreement with the displayed ab initio model.
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Fig. 9. SAXS analysis and modeling of BnC. (A) Processed solution scattering pattern of B. nigra Cruciferin (blue) in arbitrary intensity units and the fit with B. napus 11S globulin (ProCruciferin; PDB ID: 3KGL) (red) as evaluated by CRYSOL (χ2 = 1.34). (B) Kratky plot of the scattering data verifying an overall compact and rigid folding. . (C) Corresponding distance distribution functions p(r).
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Fig. 10. SAXS analysis and modeling of BnC. Ab initio model of Cruciferin with P3 symmetry (grey spheres) and B. napus 11S globulin (PDB ID: 3KGL) were superimposed.
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Table 4. SAXS data collection and analysis parameters
Data collection parameters Values X-ray source; place of data collection PETRA III; EMBL beamline P12 Wavelength (nm) 0.124 Range of s (nm-1) 0.03-4.80 Exposure time (s) 0.045 Sample-detector-distance (m) 3.1 Concentration range (mg ml-1) 2-12 Temperature (K) 283 Structural parameters
RG (nm) [based on the guinier approximation] 4.24 ± 0.25
RG (nm) [based on the ab initio model] 4.19
Dmax (nm) [based on the p(r) function] 14.0 Molecular weight determination MW from DAMMIF volume (kDa) 190 Calculated MW from sequence (kDa) 185 Software employed Initial processing of scattering intensities SASFLOW/PRIMUSQT Ab initio modeling DAMMIF Calculation and comparison of scattering data CRYSOL
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3.2.2 Identification of Eruca sativa Cruciferin (EsC)
Second Cruciferin was identified from seeds of Eruca sativa by LC-MS/MS mass spectrometry. Seven random fragments were obtained as given in (Table 5).
Table 5. LC-MS/MS spectroscopic produced amino acid sequences of EsC.
Sr. No. Peptide Sequences 1 AGVSVSRVIIEQGGLYLPTFFSSPKISYVVQGMGISGRVV (40) 2 LAGNNPQGGSQQQQQQQQNMLSGFDPQVL (29) 3 ADVYKPNLGRVTSVNSYTLPILQYIR (26) 4 GILQGNAMVL (10) 5 QVVNDNGQNVLDQQVQK (17) 6 ISFKTNANAMVSTLAGR (17) 7 LRALPLEVITNAFQISLEEA (20)
The obtained amino acid sequence of Cruciferin was used to find homologue proteins deposited in the UniProtKB database. A BLAST search was performed to trace putative similarities between the amino acid sequences of homologue protein from Raphanus sativus. The peptide sequences obtained by LC-MS/MS mass spectrometric analysis showed 63 % sequence identity with Cruciferins (CRU1) of Raphanus sativus and Brassica napus (CRU3) as shown in (Fig. 11).
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Fig. 11. Sequence alignments of EsC to the others plant Cruciferins; Cruciferin (CRU1) from Raphnus sativus and Cruciferin (CRU3) from Brassica napus, indicating a high level of residual identity (63 %). Identically conserved sequences are represented by Asterisks (*) under sequences while conserved substitutions and semi-conserved substitutions are represented by double dots (:) and single dots (.) respectively.
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3.2.2.1 Purification of EsC
The Cruciferin protein was precipitated by 30 % (w/v) ammonium sulfate from crude extract of Eruca sativa in 20 mM Tris buffer of pH 7.5. The dissolved precipitates were extensively dialyzed to remove any further salt traces and subjected to size-exclusion chromatography to obtain the purified protein fractions. Ultimately, an optimized combination of ammonium sulfate precipitation along with chromatographic steps provided a more than 95 % pure Cruciferin solution from seeds of Eruca sativa and was further verified by SDS-PAGE analysis. Purified Cruciferin protein was subjected to size exclusion chromatography using Hi-Load 16/60 Superdex 200 (GE healthcare) column to know the correct molecular weight of the protein as shown in (Fig.12A). The SDS-PAGE analysis of the purified protein bands under both reduced and non-reduced conditions indicated the presence of inter-chain disulfide linkage as shown in (Fig. 12B).
Fig. 12. Purification and SDS-PAGE analysis of EsC (A) A highly purified EsC was obtained after gel filtration (Hi-Load 16/60 Superdex 200 column) chromatography. (B) 12 % SDS-PAGE of the purified Cruciferin from E. sativa fractions. Lane M contains molecular weight standards. Lane 1; purified Cruciferin (50 kDa approximately highlighted in red box) without mercaptoethanol; Lane 2: under reduced form in presence of mercaptoethanol showing two bands of 30 and 22 kDa also known as α and β-polypeptides respectively.
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3.2.2.2 Secondary structure determination of EsC CD spectroscopy data shows the presence of both α-helix and β-sheet conformation in the native fold of EsC. The course of the averaged spectrum is typical for proteins containing α-helix and β-sheet structure (Manavalan et al., 1983). (Fig. 13) shows the CD spectrum resulting in a calculated content of about 7.5 % α-helix, 47.6 % β-sheet, 7 % β-turn and 37.9 % not compactly folded (random), along with an RMS value of 6.9 for the respective reference fit curve.
Fig. 13. Averaged far-UV CD spectrum of seed EsC. Although CD spectra is indicating the presence of both helix and sheets, however, the major secondary structure is β-sheeted followed by nonstructural or loop areas.
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3.2.2.3 Dynamic Light Scattering analysis of EsC
The homogeneity of purified EsC was checked applying the dynamic light scattering (DLS). Different buffers were used to optimize the conditions for most homogeneous and monodisperse protein solution. For this purpose, the purified solution (41 mg/ml) was centrifuge at 17,000 g for one hour in ultracentrifuge in order to remove all larger aggregates and further impurities were removed by the help of 0.22 µm syringe filter. (Fig. 14A) is showing a highly monodisperse EsC solution and the corresponding histogram (Fig. 14B) indicated a hydrodynamic radius of 5.5 ± 0.3 nm which is a reflection of either trimeric or hexameric nature of native EsC molecules in the solution.
The RH value of 5.5 nm provided further information about the globular shape of EsC.
Fig. 14. Dynamic light scattering (DLS) measurement of E. sativa native Cruciferin (EsC). (A) is showing the monodisperse nature of Cruciferin solution at a concentration of 41 mg/ml. (B) is showing a hydrodynamic radius (RH) of 5.5 ± 0.36 nm confirming the trimeric nature with molecular weight of approximately 170 kDa for the real molecule.
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3.2.2.4 Crystallization and Diffraction data analysis of EsC
Almost 500 different crystallization conditions (Pact, JCSG, Morpheus and Ammonium Sulfate suites) were screened to crystallize the EsC at a concentration of 41 mg/ml. Initial crystals were obtained from condition E11 of Morpheus (Molecular Dimensions, UK) containing 10 % PEG 4,000, 20 % (v/v) glycerol and 0.1 M trizma base (pH 8.5) and 0.03 M of each Diethyleneglycol, triethyleneglycol, tetraethyleneglycol and pentaethyleneglycol compounds. This condition was optimized further through hanging drop method and a drop consisting of 2 µl of the protein solution and 2 µl of the reservoir solution were equilibrated against 1ml of reservoir solution in linbro plates. Single crystals of approx. (0.1 × 0.05 × 0.05 mm) sizes were appeared after 10 days, as shown in (Fig. 15). Unfortunately, crystals of EsC diffracted up to a maximum resolutions of 7 Å, which was unsuitable for structure determination and therefore, the data collection was stopped after processing of few images.
Fig. 15. In situ microscopic analysis of EsC crystals with and without UV light. (A) is showing the small sized Cruciferin crystals (0.1 × 0.05 × 0.05 mm) under visible microscope while (B) is showing the same crystals producing fluorescence under UV light.
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3.2.2.5 SAXS data analysis of EsC
The SAXS data of EsC from E. sativa showed that it is exist in trimeric state in 20 mM Tris-HCl buffer of pH 7.5. The average scattering is characterized by radius of gyration (Rg) 4.3± 0.30 nm according to the guinier approximation determined by AUTORG implemented in PRIMUS (Petoukhov et al., 2012) and the maximum dimension Dmax obtained from the collected data were 15.0 ± 0.41 nm, respectively (Fig 16A and 16B). Based on the shape of the Kratky plot curve, the protein is rather rigid and compactly folded (Fig. 16B). A tentative ab initio model of EsC (Fig. 17, grey spheres) displays an extended ―loop structure‖ sticking out of the nearly globular surface in agreement with the p(r) function (Fig. 16C). The minimized χ2-value of 1.41 calculated from average scattering pattern of EsC in comparison to that of ab initio DAMMIF (Franke & Svergun, 2009) confirms the presence of an extended terminus or loop of EsC that possesses some flexibility in solution. Considering the symmetry of a putative homotrimer, ten ab initio models were calculated using DAMMIF sharing a mean NSD value of 0.957 ± 0.271 (Fig. 17). The displayed ab initio dummy model with P3 symmetry was obtained. The shape factor (calculated by dividing radius of gyration Rg over hydrodynamic radius RH) has a value of 0.76 which is indicative for a nearly globular particle in agreement with the displayed ab initio model.
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Fig. 16. SAXS analysis and modeling of EsC. (A) Processed solution scattering pattern of EsC (green) in arbitrary intensity units and the fit with B. napus 11S globulin (ProCruciferin; PDB ID: 3KGL) (blue) as evaluated by CRYSOL (χ2 = 2.0). (B) Kratky plot of the scattering data verifying an overall compact and rigid folding. (C) Corresponding distance distribution functions p(r).
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Fig. 17. SAXS analysis and corresponding ab initio model of EsC with P3 symmetry (grey spheres) and 3KGL have been superimposed. It indicated that native EsC molecules exist in trimeric form possessing a molecular weight of approximately 180 kDa.
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3.3 Ubiquitin
Ubiquitin protein is small and globular protein and present in all eukaryotic cells and also present even in the prokaryotic cell. The amino acids sequence is highly conserved in all organisms. Ubiquitin protein was isolated, identified and purified from Sisymbrium irio seeds
3.3.1 Protein purification, identification and amino acid sequencing
Ubiquitin protein, isolated from seeds of S. irio, was purified by subjecting to size- exclusion chromatography in 100 mM citrate and 150 mM NaCl buffer, pH 3.0 to obtain the purified protein fractions. (Fig. 18A) shows the presence of three protein bands appeared at pH 3.0 while two distinct peaks obtained in the chromatogram as displayed in Fig. 18B. Pure protein fractions showing high absorbance at 280 nm were stored at 4 °C. One dimensional SDS-PAGE was performed under both reducing (with β- mercaptoethanol) and non-reducing (without β-mercaptoethanol) conditions to investigate the molecular weight and number of protein bands in S. irio extract as demonstrated in Fig. 18C. The results of SDS-PAGE revealed that the purified protein has a molecular weight of approximately 17 kDa under non-reducing conditions (lane L1) and about 9 kDa under reducing conditions (lane L2) in (Fig. 18C). These results suggest the possibility of the presence of a dimer of the purified protein in solution. The purified protein was in-gel digested with trypsin and resulting peptides were analyzed by LC–MS/MS mass spectrometry. Five peptides are represented in the (Table 6).
Table 6. LC–MS/MS mass spectroscopic produced amino acid sequences of SiU.
Sr. No. Peptide Sequences 1 KTITLEVESS (10) 2 DTIDNVKAK (9) 3 LIFAGKDL (8) 4 EDGRT LADYN (10) 5 IDKESTLHLVLRLR (12)
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These identified protein peptides of interest from the S. irio seeds which on BLAST search confirmed the ubiquitin identity as exemplified in (Table 7). IEF showed single band on the IEF gel revealing the basic pI of 9.2 as illustrated in (Fig. 18D).
Table 7. Sequence similarity of identified peptide fragments obtained from LC–MS/MS.
Protein name, organism & % Peptide fragments E-value UniProtKB / Swiss-Prot ID: Identity (Amino acid numbering) polyubiquitin, Arabidopsis KTITLEVESS 90 4 x 10-3 thaliana, Q39256.1 (89-98) polyubiquitin, Arabidopsis DTIDNVKAK 100 2.0 x 10-2 thaliana, Q39256.1 (99-107) polyubiquitin, Caenorhabditis LIFAGKDL 88 8.4 elegans, P0CG71.1 (119-126) polyubiquitin, Arabidopsis EDGRTLADYN 100 7.0 x 10-4 thaliana, Q39256.1 (129-138) polyubiquitin, Caenorhabditis IDKESTLHLVLRLR 93 3.0 x 10-6 elegans, P0CG71.1 (137-150) KTITLEVESS Ubiquitin, Triticum aestivum or 77 1.0 x 10-24 bread wheat, P69326.2 (11-74) DTIDNVKAK
LIFAGKDL Ubiquitin, Avena sativa or oat, 77 1.0 x 10-24 EDGRTLADYN P69310.1 (11-74)
IDKESTLHLVLRLR
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Fig. 18. Extraction, purification and isoelectric focusing of SiU. (A) SDS-PAGE analysis of S. irio crude extract at pH 3.0 in lane L, (B) Size-exclusion chromatogram obtained from Superdex 200 column, (C) SDS-PAGE analysis of SiU protein, comprising lane M for molecular weight standard, lane L1 for non- reducing condition and lane L2 for reducing condition. (D) Isoelectric focusing (IEF) of purified SiU showing single band on strip with the alkaline pI of 9.2.
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3.3.2 Characterization in solution of SiU
3.3.2.1 CD spectroscopy
Fresh samples were dialyzed at a low salt concentration buffer and secondary structure was determined using circular dichroism (CD) spectroscopy. The shape of the averaged spectrum is typical for proteins containing α-helix and β-sheet structure possessing two distinct minima at 195 nm and 260 nm. (Fig. 19) has shown the CD spectrum resulting in a calculated content of about 6.5 % α-helix, 39.3 % β-sheet, 0.7 % turn, 53.5 % random coil. The root-mean-square (RMS) values between the fitted curves (Reed & Kinzel, 1984) and the Ubiquitin data was found as 5.11 %.
Fig. 19. Circular dichroism of SiU showed Far-UV CD spectrum that consist of high content of 39.3 % β- sheet.
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3.3.2.2 DLS measurements
The SiU protein sample was subjected to DLS measurements after centrifugation, in order to remove unordered aggregates. (Fig. 20A) was shown the DLS results of SiU protein revealing the monodispersive nature in solution, inferred from their single line signal with the hydrodynamic radius (RH) of 2.44 ± 0.37 (Fig. 20B). Although Ubiquitins have also been reported as 9 kDa in native conformation as well, however, SiU appeared in dimeric form of 17 kDa under non-reduced conditions of SDS-PAGE. This RH value of 2.44 provided further confirmation of the dimeric status of globular shape SiU in solution form.
Fig. 20. Dynamic light scattering of Ubiquitin (A) DLS and (B) the hydrodynamic radius (RH) of purified SiU.
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3.3.2.3 In-Silico analysis of SiU
(Fig. 21) demonstrates the multiple sequence alignment of S. irio Ubiquitin (SiU) amino acid sequence with other homologues Ubiquitin sequences present in the UniProtKB database. The analysis showed that the identified 51 amino acid sequence of SiU possess 77 % identity with other Ubiquitin sequences obtained from different species; e.g., Camelus dromedaries, Caenorhabditis elegans, Acanthamoeba castellanii, Trypanosoma cruzi, Cryptococcus neoformans, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Neurospora crassa, Coprinellus congregates, Chlamydomonas reinhardtii, Daucus carota, Deschampsia antarctica, Oryza sativa, Nicotiana sylvestris, Arabidopsis thaliana, Helianthus annuus, Brassica rapa, Triticum aestivum, Avena sativa and Hordeum vulgare. Minor changes were observed in SiU sequence at positions 42, 49 and 62. Structure and sequence of Ubiquitin was recently reviewed in the literature which revealed that despite of few conservative changes; Ubiquitin sequence is almost invariant from yeast to man (Aguilar & Wendland, 2003). Presence of alanine residues in place of serine at positions 28 and 57 were considered as conservative changes because it was also observed in other sequences. However, replacement of R42 by K42, Q49 and Q62 by D49 and D62, respectively are non-conserved changes and might be consider as the unique features of SiU. The arranged amino acid sequence (1-51) of SiU was perfectly aligned with the amino acids (1-76) of Saccharomyces cerevisiae Ubiquitin (Sc-Ub2, PDB-ID: 4NNJ_D). The alignment induces two gaps in the SiU sequence corresponding to the amino acids # 1-10, and 29-41 of Sc-Ub2.
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Fig. 21. Multiple sequence alignment of Ubiquitin proteins from different species present in UniProtKB database. Black background showed overall conserved regions and grey background also revealed conserved regions but not obtained through LC–MS/MS in SiU sequence, - represents gaps whereas * indicates exactly identical residues. Red boxes show conserved changes while green boxes represent non-conserved changes.
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3.3.2.4 Quality of the 3D-structure model
A homology model of SiU with amino acids (11-28 and 42-76) was successfully generated by using the crystal structure information of Sc-Ub2 (PDB-ID: 4NNJ_D). The quality of the model was assessed by Procheck online tool which gave a Ramachandran plot for the modeled SiU as displayed in (Fig. 22). Out of 51 modeled residues, Ramachandran plot showed 95.7 % in the most favored region, 2.1 % in allowed region, 2.1 % in generously allowed and 0 % in disallowed region
Fig. 22. Ramachandran plot of the modeled SiU structure.
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.3.3.2.5 Structure comparison of SiU to Human Erythrocytic Ubiquitin (heu)
The modeled SiU structure is good enough for further analysis. The crystal structure of human erythrocytic Ubiquitin (heu) has been explained in detail and its structural features can be compared with the modeled SiU (Vijay et al., 1987). In general, Ubiquitin structure is compact in nature and only last six C-terminal residues (71-76) are unordered. (Fig. 23A) displays the crystal structure of heu showing all of its structural features. According to the human erythrocytic Ubiquitin numbering (PDB-ID: 1UBQ), Ubiquitin structure comprised of five β-strands holding residues 1-7, 11-17, 40-45, 48-50, and 65-72 (Fig. 23A, orange sheets). Furthermore, one α-helix comprising of residues 23- 34 and one 3-10 helix consisting 56-59 residues (Fig. 23A, sea green and light blue helices). The most important feature of Ubiquitin is its seven lysines (K6, K11, K27, K29, K33, K48 and K63) which cover all surfaces of Ubiquitin and point into distinct directions (Fig. 23A, brown ball & sticks). K6 and K11 are located in the most dynamic region of Ubiquitin that may undergo conformational changes upon association with Ubiquitin binding domains (UBDs) (Fig. 23A). As K27 is buried, linkage assembly through this residue would require localized changes in Ubiquitin structure (Geng & Tansey, 2012). A hydrophobic surface comprising Q2, F4, and T12 (F4 patch, Fig. 23A, yellow surface) is obliged for cell division in yeast (Sloper-Mould et al., 2001).
This F4 patch might function in trafficking and interacts with the UBAN domain (Rahighi et al., 2009) and the Ubiquitin-specific protease (USP) domain of deubiquitinating enzyme (DUBs) (Hu et al., 2002). In higher eukaryotes, a 3D motif that includes T12, T14, E34, K6, and K11 (TEK-box, Fig. 38A, orchid surface), is necessitate for mitotic degradation (Jin et al., 2008). A hydrophobic surface, centered on I36, L71 and L73 (I36 patch, Fig. 38A, salmon surface) can mediate interactions between Ubiquitin molecules in chains. It is recognized by HECTE3s (Kamadurai et al., 2009), DUBs (Hu et al., 2002) and Ubiquitin binding domains (UBDs). Ubiquitin is often recognized through a hydrophobic surface, comprising of I44, L8, V70, and H68 (I44 patch, Fig. 23A, cyan surface) which is bound by the proteasome and most UBDs, rendering it vital for cell division (Dikic et al., 2009; Shih et al., 2000; Sloper et al., 2001). Further, the β1/β2 loop containing L8 shows flexibility that is essential for recognition by Ubiquitin-binding proteins (Lange et al., 2008). The modeled structure of SiU is explained in comparison
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Fig. 23. Structural features of SiU. (A) Human erythrocytic Ubiquitin (PDB-ID: 1UBQ) and (B) S. irio Ubiquitin (SiU). Structural motifs include F4 patch yellow, TEK box orchid, I36 patch salmon, and I44 patch cyan in colors.
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3.4 Napin
Napin (2S albumin) is a small molecular weight protein present in the seeds of different plants including Brassicaceae as well. It is second abundant seed storage protein after Cruciferin in Brassicaceae. It has the molecular weight ranges between 12 to 16 kDa. An approximately 16 kDa Napin (BnN) has been identified and characterized from seeds of Brassica nigra
3.4.1 Extraction and identification of BnN
Napin was extracted from 10 g finely grinded seed powder in 100 ml of 20 mM sodium acetate buffer of pH 5.5 at room temperature. After stirring and centrifugation, the transparent crude extract was loaded on SDS-PAGE under reduced condition and was subsequently blotted on PVDF for N-terminal amino acid sequence. A primary sequence of 15 amino acids (IPPXRREFQQAXHL) was obtained through Edman degradation. Analysis of this sequence via the PROTEIN BLAST (UniProtKB) showed a maximum homology with other known Napins of the Brassicaceae family as shown in (Table 8).
The sequence showed 78.8 % amino acid identity to that of Napin (2S albumin) from Raphanus sativus and BnN also showed 71.4 % to Napin-3 and Napin from Brassica napus and Brassica campestris respectively.
Table 8. N-terminal sequence comparison showed high homology (78.8%) with R. sativus Napin (2S albumin) and similarly with others Brassicaceae.
Plants Proteins UniProtKB ID: Sequences Identity (%)
B. nigra Napin IPPXRREFQQAXHL
Napin (2S R sativus Q9S8Y8 IPRCRREFQQAQHL 78.8 albumin)
B. napus Napin-3 P80208 IPKCRKEFQQAQHL 71.4
B campestris Napin Q7DMU4 IPKCRKEFQQAQHL 71.4
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3.4.2 Purification of BnN
The B. nigra Napin (BnN) protein was stepwise purified by an optimized combination of cation and size exclusion chromatography which provided ample quantities of highly purified protein. Initially, 95 % ethanol was used to remove the oily nature of the seeds. The 30 % ammonium sulfate precipitation had already removed the 12S globulins/Cruciferin (BnC) as pellet (described earlier) while 2S albumin and other contaminating proteins remained in the supernatant. The supernatant was dialyzed against same extraction buffer (20 mM acetate buffer, pH 5.5) and loaded on Cation exchanger column Mono S 5/50 GL (GE Healthcare). The column was pre-equilibrated with acetate buffer A. After sample loading, a linear gradient of 1-100 % buffer B (20 mM acetate, pH 5.5 and 1 M NaCl) was applied and BnN started to elute at 20 % gradient. The corresponding pure fractions were pooled after SDS-PAGE analysis. The pooled protein was further subjected to size exclusion chromatography using Hi Load 16/60 Superdex 75 in order to attain highly pure BnN (Fig. 24A). The purified BnN showed signal band with a molecular weight of approximately 16kDa on 15% SDS-PAGE under non-reduced form (without mercaptoethanol) and in reduced form (with mercaptoethanol) showed the presence of inter chain disulfide linkage as shown in lanes 1and 2 of (Fig. 24B).
Fig. 24. Purification and SDS-PAGE analysis of BnN from Brassica nigra. (A) is Indicating Size-exclusion chromatogram obtained from Superdex 75 column. (B) 15% SDS-PAGE analysis of B. nigra purified protein (BnN); lane M is showing for molecular weight standard, lane L1 is showing the intact 16 kDa BnN under non-reducing condition and lane L2 is showing the reduction of 16 kDa into smaller sub-unit of approximately 10 kDa while smaller sub-unit of 6 kDa is not visible.
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3.4.3 Characterization in solution
3.4.3.1 CD spectroscopy
Secondary structure of (BnN) was determined in 20 mM sodium acetate buffer pH 5.5 by using circular dichroism (CD) spectroscopy. CD spectroscopy data shows the presence of both α-helix and β-sheet conformation in the native fold of BnN. The course of the averaged spectrum is typical for proteins containing α-helix and β-sheet structure (Manavalan et al., 1983), exhibiting two distinct minima at 210 nm and 225 nm. (Fig. 25) shows the CD spectrum resulting in a calculated content of about 38 % α-helix, 20 % β- sheet, 11 % turn and 31 % compactly folded (random), along an RMS value of 3.26.
Fig. 25. CD spectra indicating the secondary conformation of Brassica nigra Napin (BnN). Although BnN is composed of both helices and sheets, however, the major secondary structure is α-helix and is compactly folded.
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3.4.3.2 DLS measurement of BnN solution
The BnN was subjected to DLS measurements after centrifugation, in order to remove unordered aggregates. (Fig. 26A) is showing the DLS results of BnN revealing the monodispersive nature in solution, inferred from their single line signal. The intact 16 kDa
BnN showed hydrodynamic radius (RH) of 1.99 ± 0.1 nm and in comparison to standard hydrodynamic radii of globular proteins, it confirms the presence of a monomeric state in solution (Fig. 26B).
Fig. 26. Dynamic light scattering (DLS) measurements of BnN. (A) is confirming a monodisperse solution of BnN at a concentration of 15 mg/ml. (B) is indicating the hydrodynamic radius (RH) (2.0± 0.1 nm) of purified BnN.
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3.4.3.3 Effect of temperature on BnN monodispersity
At a concentration of 15 mg/ml, BnN was exposed to two different temperatures (4 and 20 °C) in order to optimize the crystallization conditions. The BnN stability or monodispersity was observed carefully for three continuous days at 4 °C in 20 mM acetate buffer of pH 5.5. Effect of low temperature treatment on the stability is shown in the (Fig. 27). DLS data of 4 °C indicated that BnN is rather more stable and monodisperse protein and corresponding hydrodynamic radius (2.1± 0.2) was also constant.
3.4.3.4 Effect of 20 ºC on BnN stability
Similarly, the stability of BnN was monitored at 20 °C for three continuous days with same constant buffering conditions. Effect of 20 °C temperature on the stability is shown in the (Fig. 28). A similar stability trend was experienced for BnN at 20 °C indicating no aggregation or development of heterogeneity inside the protein solution during the period of three days similar to that of 4 °C indicating a more stable and constant monomeric conformation of the molecule.
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Fig. 27. DLS measurements are showing the effect of 4 ºC temperature on the monodispersity of the BnN for three continuous days. A1, B1 and C1 are showing the monodisperse stability of BnN at first, second and third day respectively. Similarly, A2, B2 and C2 are showing the corresponding hydrodynamic radii (2.16± 0.3) for three consecutive days with zero fluctuation.
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Fig. 28. DLS measurements are showing the effect of 20 ºC temperature on the monodispersity of the BnN for three continuous days. A1, B1 and C1 are showing the monodisperse stability of BnN at first, second and third day respectively. Similarly, A2, B2 and C2 are showing the corresponding hydrodynamic radii (2.16± 0.3) for three consecutive days with zero fluctuation.
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3.4.4 Crystallization of BnN
Crystallization of BnN at a concentration of 15 mg/ml was performed in 480 different crystallization conditions (Compass, Pact, JCSG, Morpheus and Ammonium Sulfate suites). Small size (35 µm) diamond shape crystals (Fig. 29) appeared after two months in A2 (0.1 M Sodium citrate, pH 5.5, 20 % w/v PEG 3k) screening condition of JCSG Suite. Optimization of BnN micro crystals was performed in linbro plates by using hanging drop method. However, no significant difference of crystal size was obtained and micro crystals diffracted to low resolution (9 Å) which was not suitable for structure solution and further processing was stopped.
A B
Fig. 29. In situ microscopic analysis of BnN micro crystals. (A) is showing the small sized Napin crystals (0.035 × 0.02 × 0.018 mm) under visible microscope while (B) is showing the zoom in view of the same crystal.
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3.4.5 Bioactivity assays analysis
3.4.5.1 BnN antifungal activity
Anti-fungal activity of BnN against filamentous fungi Fusarium solani and Fusarium oxysporum was assessed by applying the disc diffusion method. Two quantities of BnN i.e. 15 µg and 30 µg/disc were applied to observe the fungal growth inhibition. (Fig. 30A and 30B) is clearly showing that 30 µg of BnN significantly inhibited the growth of both Fusarium solani and Fusarium oxysporum on potato dextrose agar.
Fig. 30. Inhibitory activity of BnN towards Fusarium solani (A) and Fusarium oxysporum (B). [a: Topsin® fungicide (20 µl/disc); b: 30 µl/disc 20 mM acetate buffer (pH 5.5); c: Diluted BnN protein (15 µg/disc); d: Concentrated BnN protein (30 µg/disc)].
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3.4.5.2 Entomotoxic assessment
Purified Napin protein (BnN) extracted from the Brassica nigra seeds significantly inhibited the T. castaneum growth. Three different doses were used and different biological traits e.g., number of larvae and pupae as well as total adult populations were monitored. Results clearly indicated the effectiveness of BnN flour mixture when fed to T. castaneum. In comparison to control, all protein concentrations produced significant reduction of population growth. The highest larval population was observed in the control group i.e. (126.8 ± 7.2) while all protein concentrations reduced the larval population significantly (P < 0.0006, F = 9.96) along maximum mortality (12.0 ± 4.3) at 3 mg/ml. Similarly, the total number of pupae and adults was strongly decreased at all BnN concentrations in comparison to control experiment. Comparison between treatments indicated the highest mortality for all three populations (average larval, pupal and adult) at 3 mg/ml concentration followed by 2 mg/ml and 1 mg/ml. Thus 3 mg/ml concentration of BnN is the most effective dose in reducing T. castaneum population as shown in (Table 9).
Table 9. Entomotoxin activity of BnN against different life stages of T. castaneum. Three different Napin concentrations (1, 2 and 3 mg/ml) were used to see the insecticidal efficacy. Control was comprised of 20 mM acetate buffer of pH 5.5.
BnN (mg/ml) No. of Larvae No. of Pupae No. of Adults
1.0 15.6 ± 5.8b 4.4 ± 1.3b 1.0 ± 0.7b
2.0 15.2 ± 5.7b 3.4 ± 1.6b 2.0 ± 1.5b
3.0 12.0 ± 4.3b 1.8 ± 0.8b 0.8 ± 0.5b
Control 126.8 ± 7.2a 62.2 ± 12.8a 26.2 ± 6.1a
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3.5 Thionin
Thionins are low molecular weight proteins of about 15 kDa that are composed of Signal peptide, Thionin and Acidic domain parts. After processing, mature Thionin is a Cys-rich protein of approximately 5 kDa and are famous for multiple disulfide linkages that lead towards the development of dimers or trimers. In higher plants, Thionins act as pathogenesis-related proteins (PRs) as a part of plant defense mechanism. A 15 kDa Probable Thionin protein, comprising of Signal peptide, Thionin and Acidic domain parts; was identified and purified from seeds of Eruca sativa locally known as ―Tarameera‖.
3.5.1 Extraction and identification of pEsT
Precursor Eruca sativa Thionin (pEsT) was extracted from seeds in 0.1 M sodium phosphate buffer of pH 7.0 and was identified by determining its fragmented primary sequence. The protein was digested with trypsin and resulting peptides were analyzed by MALDI-TOF/TOF mass spectrometry. Four peptides were identified and are presented in (Table 10).
Table 10. Random fragmented amino acid sequence of pEsT obtained through MALDI- TOF/TOF mass spectrometry. In total four fragments comprising of 58 residues were obtained.
Sr. No. Peptide Sequences 1 GKTLIVSVLIMSLFM (15) 2 PSIQARTFYNACLF (14)
3 TEYCKLGCV (10) 4 VCGALTILQNSDAS (15)
The amino acid sequence of pEsT was used to find homologue proteins already deposited in the UniProtKB database. A BLAST search was performed to trace putative similarities between the amino acid sequences of homologue proteins. Further analysis showed that fragments of Thionin have the highest similarity with Thionin 2.4 from Arabidopsis thaliana. The peptide sequences obtained by mass spectrometric analysis showed 100 % sequence identity with Thi 2.4 as shown in (Fig 31).
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Fig. 31. Sequence homology of precursor Eruca sativa Thionin (pEsT) to Thi 2.4 from Arabidopsis thaliana. Symbol ―X‖ is representing the gaps between the obtained peptides.
A relatively low homology was observed with THN of Brassica rapa showing 32 % sequence. Fig. 32 is showing the alignment of the pEsT fragmented sequence with other plant Thionins. The six cysteines at positions 3, 4, 27, 33, 44 as well as an arginine at position 10 are shown in red color and an aromatic residue at position 13 are well conserved as highlighted with green color (Fig. 32).
Fig. 32. Sequence alignments of precursor Eruca sativa Thionin (pEsT) to other plant Thionins. Thionin (Thi 2.4) from Arabidopsis thaliana and Thionin from Brassica rapa. (THN).
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3.5.2 Purification of pEsT
Thionin (pEsT) was precipitated by 75 % (w/v) ammonium sulfate from the crude extract of Eruca sativa. The dissolved precipitates were extensively dialyzed to remove any further salt traces and subjected to size-exclusion chromatography to obtain the purified protein fractions. Ultimately, an optimized combination of ammonium sulfate precipitation along with chromatographic steps provided a more than 95 % pure Thionin solution from seeds of Eruca sativa, as judged by SDS-PAGE analysis. Size-exclusion chromatography was performed in 25 mM sodium phosphate, pH 7.0 and 150 mM NaCl. (Fig. 33A) showed two distinct peaks (PI and PII) corresponding to Cruciferin and pEsT respectively. pEsT rich fractions with the highest absorbance at 280 nm were pooled and stored at 4 °C. One dimensional SDS-PAGE was performed under both reducing and non- reducing conditions to analyze the banding pattern as shown in (Fig. 33A). The results of SDS-PAGE showed that Thionin has a molecular weight of approximately 16 kDa under non-reducing conditions (lane 1 of SDS-PAGE), but under reducing conditions (lane 2), two bands appeared on the gel. A 16 kDa Thionin is reduced in approximately 11 kDa and 5 kDa bands as shown in Lane 2 (Fig. 33B). The single band on the IEF gel reveals the basic pI of 8.2.
Fig. 33. (A) Purification of pEsT by calibrated size exclusion chromatography (HiLoad 16/60 Superdex 75) subsequent to ammonium sulfate precipitation. The absorbance at a wavelength of 280 nm is displayed and the absorbance peak of pure Thionin with a retention volume of 80.7 ml corresponding to approximately 20.3 kDa is labeled. (B) SDS-PAGE analysis of pEsT under non-reducing conditions (lane 1) and in the
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Chapter 3 Results presence of 20 mM DTT (lane 2). Upon addition of DTT the 16 kDa pre-Thionin is split into domains of approximately 11 and 5 kDa respectively.
3.5.3 Characterization in solution
3.5.3.1 Secondary structure determination of pEsT
CD spectroscopy data indicates the presence of both α-helix and β-sheet conformation in the native fold of pEsT. The course of the averaged spectrum is typical for proteins containing α-helix and β-sheet structure (Manavalan et al. 1983), possessing two distinct minima at around 210 nm and 221 nm. (Fig. 34) shows the CD spectrum resulting in a calculated content of about 38 % α-helix, 9 % β-sheet, 19 % turn, 34 % not compactly folded (random), along with an RMS value of 2.955 for the respective reference fit curve
Fig. 34. Far-UV CD spectrum of pEsT from E. sativa as an average of 10 individual spectra in 25 mM phosphate buffer at pH 7.0. Spectra indicate that secondary structure of Thionin has major part of α-helix and two distinct minima at around 210 nm and 221 nm.
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3.5.3.2 Dynamic Light Scattering analysis of pEsT
A monodisperse Thionin solution in 20 mM phosphate buffer of pH 7.0 showed a hydrodynamic radius of 2.5 ± 0.2 nm as shown in (Fig. 35). Blue peak is showing relatively higher RH radius of 16 kDa precursor molecule comprising of 11 kDa acidic part and 5 kDa probable Thionin linked together through disulfide linkage. However, after the addition of Dithiothreitol (DTT) to the precursor molecule, a shift in hydrodynamic radius was observed as shown by red peak which is clear indicative of the detachment of acidic part from probable Thionin. Additionally evidence can also be seen by broadness of the peak which is most probably representing the mixture of precursor molecule (16 kDa), 11 kDa acidic part and 5 kDa probable Thionin altogether in the solution along with DTT.
Fig. 35. The Particle size distribution obtained by dynamic light scattering reveals the monodispersity of the pEsT solution. A hydrodynamic radius (RH) of 2.5 ± 0.1 nm was determined, which would corresponding to a molecular weight of around 25 kDa, if the particle is globular. Under reducing conditions the hydrodynamic radius decreases to 1.8 ± 0.2 nm as shown by the read peak.
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3.5.3.3 Effect of temperature on pEsT stability at 4 ºC
At a concentration of 25 mg/ml, pEsT was exposed to two different temperatures (4 and 20 °C) in order to optimize the crystallization conditions. The pEsT stability or monodispersity was observed carefully for three continuous days at 4 °C in 20 mM sodium phosphate. Effect of low temperature treatment on the stability is shown in the Fig. 36. DLS data of 4 °C indicated that pEsT is rather more stable and monodisperse protein and corresponding hydrodynamic radius (2.5 ± 0.2) was also constant.
3.5.3.4 Effect of temperature on pEsT stability at 20 ºC
Similarly, the stability of pEsT was monitored at 20 °C for three continuous days with same constant buffering conditions. Effect of 20 °C temperature on the stability is shown in the (Fig. 37). A similar stability trend was experienced for pEsT at 20 °C indicating no aggregation or development of heterogeneity inside the protein solution during the period of three days similar to that of 4 °C indicating a more stable and constant monomeric conformation of the molecule.
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Fig. 36. DLS measurements are showing the effect of 4 ºC temperature on the monodispersity of the pEsT for three continuous days. A1, B1 and C1 are showing the monodisperse stability of pEsT at first, second and third day respectively. Similarly, A2, B2 and C2 are showing the corresponding hydrodynamic radii (2.00± 0.2) for three consecutive days with zero fluctuation.
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Fig. 37. DLS measurements are showing the effect of 20 ºC temperature on the monodispersity of the pEsT for three continuous days. A1, B1 and C1 are showing the monodisperse stability of pEsT at first, second and third day respectively. Similarly, A2, B2 and C2 are showing the corresponding hydrodynamic radii (2.10± 0.3) for three consecutive days with zero fluctuation.
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3.5.3.5 Crystallization of pEsT
Purified pEsT was crystallizes at the concentration of 25mg/ml. Crystallization experiment of pEsT was performed in 480 different crystallization conditions (Compass, Pact, JCSG, Morpheus and Ammonium Sulfate suites) Small size (25 µm) crystals (Fig. 38) appeared after one and half months in D12 (0.002M Zinc chloride, PH 8.0,0.1MTris, 20 % w/v PEG 6000) screening condition of Pact. Optimization of pEsT micro crystals was performed in linbro plates by using hanging drop method. However, no significant difference of crystal size was obtained and micro crystals diffracted to low. Which was not suitable for structure solution and further processing was stopped.
Fig. 38. In situ microscopic analysis of pEsT micro crystals. (A) is showing the small sized pEsT crystals (0.025 × 0.02 × 0.022 mm) under visible microscope while (B) is showing the optimization Pact D12 condition in Linbro plate. Again micro crystals were obtained and diffracted to low.
3.5.3.6 Assessment of tertiary and quaternary structure
The average scattering is characterized by an Rg of 1.96 ± 0.01 nm according to the guinier approximation determined by AUTORG implemented in PRIMUS (Petoukhov et al., 2012). The P(R) function is indicative for a prolate particle with a maximum diameter of 6 nm (Fig. 39A). Based on the shape of the Kratky plot curve the protein is rather rigid and compactly folded (Fig. 39B).According to the volume of correlation, the molecular weight of Thionin is approximately 15.5 kDa. Considering the symmetry of a putative homotrimer, ten ab initio models were calculated using DAMMIF (Franke and Svergun, 2009), sharing a mean NSD value of 1.633 ± 0.238 (Fig. 39C). One representative model
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Chapter 3 Results of ten initially calculated ab initio models using DAMMIF is displayed below, considering P1 symmetry. The volume of the ab initio model is 31100 nm3, corresponding to approximately 15.5 kDa (Fig. 39C).
Fig. 39. (A) Small-angle X-ray scattering intensity plot of pure pEsT and the corresponding calculated fit curve (red) resembling a single ab initio model as displayed in panel C. The plot shows the dependency of the scattering intensity I on the momentum transfer s. (B) Kratky plot of the scattering intensity distribution indicating a compact relatively rigid and compact protein structure (C) Ab initio model calculated by DAMMIF possessing P1 symmetry; the scale bar is 1 nm in length.
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3.5.4 Bioactivity assays analysis
3.5.4.1 Cell cytotoxicity assessment of pEsT
Cytotoxicity of pEsT was assessed by measuring mitochondrial succinic dehydrogenase in living cells converts the MTT substance in purple formazan crystals that are insoluble in water. The number of living cells is represented by the absorbance of soluble farmazan in the visible light spectrum (Mosmann, 1983). The cytotoxic effect is shown in (Fig. 40). The most marked outcome was attained, with the highest concentration of Thionin at 15.63 µM and LC50, i.e. the dose lethal for 50 % of the cell killing obtained from the assay was in between of 12.5 µM and 15.63 µM.
Fig. 40. Effect of different concentrations of pEsT on Huh-7 cells visualized by plotting the cell viability (%) versus concentration (µM).
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3.5.4.2.1 Antifungal activity of PEsT by Disc Diffusion Method
Purified pEsT showed the anti-fungal activity against Fusarium solani and Fusarium oxysporum applying the disc method. In the anti-fungal assay, two quantities of pure Thionin i.e. 15 µg and 30 µg of protein were prepared to treat both fungus species. (Fig. 41A and 41B) clearly show that 30 µg of Thionin inhibit the growth of both Fusarium solani and Fusarium oxysporum on potato dextrose agar. A comparison of Thionin proteins from various plants species for their antifungal activity towards different phytopathogenic fungi is presented in (Table 11). The isolated pEsT has a molecular mass of around 16 kDa, larger than those of Thionin from Arabidopsis thaliana (15 kDa), Pennisetum glaucum (5 kDa), Viscum album (11 kDa) and Triticum aestivum (14 kDa).
Fig. 41. Inhibitory activity of pEsT towards Fusarium solani (A) and Fusarium oxysporum (B). [a: Fungicide; b: Buffer; c: Diluted protein (15 µg/disc); d: Concentrated protein (30 µg/disc)].
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3.5.4.2.2 Antifungal activity of pEsT by 96-well microtiter plate
The mean value of all eight technical replicates from all three measurements (in total twenty-four readings) was calculated. Each mean value from 24 to 120 h was adjusted by the mean value of the 0 h time point and showed in a line chart. At early time points (48 -72 h), the fungal growth is promoted by the plant protein Thionin. This is presumably due to the fact that the fungus metabolizes the protein and used it as a nutrition source such as nitrogen and carbon. During the measurement of the later time points (96 -120 h) the Thionin treated samples showed a reduction in growth compared to the BSA control (Fig. 42).
Fig. 42. Line chart with the standard deviation from 0 to 120 h growth of pEsT. Maximum fungal hypha growth was observed on different concentrations of BSA with 30 µg showing the maximum growth. This is probably, the fungi is consuming the protein for its growth. Among three used concentrations of pEsT, 50 µg showed the maximum inhibition of the fungal growth in comparison to BSA treatments. Significant growth difference could be observed between 30 µg BSA treatment with that of 50 µg pEsT after 96 hours of the recorded time period.
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Table 11. Comparison of pEsT protein with Thionins of other plant species.
MW Thionin Plant species Fungal species References (kDa)
Thionin Eruca sativa 16 F. solani, F. oxysporum This study
Arabidopsis Thionin 2.4 15 F. graminearum Asano et al., 2013 thaliana Pennisetum Chandrashekhara et PR protein-13 5 Sclerospora graminicola glaucum al., 2010 Fusarium solani, Sclerotinia Viscotoxin Viscum album 11 Giudici et al., 2004 sclerotiorum, Phytophtora infestans Triticum α-puroThionin 14 Rhizoctonia solani Oard & Rush, 2004
aestivum
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3.5.4.3 Entomotoxic assessment of pEsT
Purified pEsT protein extracted from the seeds of E. sativa significantly inhibited the development of T. castaneum populations at all stages. Different biological traits i.e. number of larvae, male and female pupae including adults and total population were monitored. Results clearly reveal the effectiveness of pEsT Thionin flour mixture when fed to T. castaneum. The results at all protein concentrations were highly significant when compared to the control experiment. The highest larval population was observed in the control group i.e. (130.8 ± 10.0) while it is obvious that all protein concentrations reduced the larval quantity significantly (P < 0.0006, F = 9.96). Similarly, the total number of counted pupae and adults was strongly reduced at all concentrations but recorded maximum in the control experiment (86.6 ± 10.5 and 41.0 ± 8.0) (P < 0.0, F = 22.5). In parallel, average larval, pupal and adults‘ populations were the smallest at 3 mg/ml concentration followed by 2 mg/ml and 1 mg/ml. Thus to adopt one concentration 3 mg/ml is most effective in reducing T. castaneum population. The ratio of male, female pupae and adults was also recorded; male population was significantly larger in number as compared to female population for all concentrations including the control experiment (Table 12).
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Table 12. Entomotoxin activity of pEsT against different life stages of T. castaneum. Three different Thionin concentrations (1, 2 and 3 mg/ml) were used to see the insecticidal efficacy. Control was comprised of 20 mM phosphate buffer, pH 7.0.
Concentratio Total Male Female Total Male Female Total n mg/ml larvae pupae pupae pupae adults adults adults
47.0 ± 14.4 ± 10.4 ± 24.8 7.4 ± 6.2 ± 13.6 1 9.6b 2.2b 2.0b ±3.8b 0.9b 0.6b ±1.4b
51.8 ± 13.0 ± 9.4 ± 22.0 4.8 ± 4.6 ± 9.4 ± 2 21.5b 4.0b 3.0b ±7.1b 1.2b 0.7b 1.9b
39.4 ± 10.8 ± 9.4 ± 22.2 6.4 ± 5.0 ± 1.4 ± 3 9.1b 1.5b 1.8b ±3.2b 0.7b 0.4b 0.9b
130.8 45.6 ± 41.0 ± 86.6±10. 23.4 17.6 ± 41.0 control ± 4.2a 7.0a 5a ±3.8a 4.7a ±8.0a 10.0a
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DISCUSSION
Chapter 4 Discussion
4.1 Brassica nigra Cruciferin (BnC)
Seed storage proteins are accumulated in protein storage vacuoles (PSVs) during seed development (Jiang et al., 2001) and are mobilized to release nitrogen and sulfur during seed germination (Müntz, 1998). A Cruciferin (50-55 kDa) from the seeds of Kali rai (B. nigra) was isolated and thoroughly characterized. The N-terminal amino acid sequence of B. nigra Cruciferins shares 82 % sequence homology with a 12S CRC (Cruciferins) from Arabidopsis thaliana. Further homology comparison suggested a high similarity to already known seed storage Cruciferins of the family Brassicaceae as shown in (Table 3). Occurrence of Cruciferins in seeds of many different species is a good indication of their wide spectrum occurrence among members of family Brassicaceae.
The B. nigra Cruciferin (BnC) with its polypeptide chains and the corresponding isoforms were analyzed by one- and two-dimensional electrophoresis techniques respectively. Cruciferin was precipitated from the crude extract of defatted powder by ammonium sulfate (30% saturation) precipitation. Excessive salt was removed by extensive dialysis and partially pure protein was subjected to gel filtration chromatography with PBS buffer pH 6.5 (Fig. 4A) in order to remove further impurities. Ultimately, an optimized combination of ammonium sulfate precipitation along with chromatographic steps provided a 95% pure Cruciferin protein (Fig. 4B) from seeds of B. nigra. One dimensional SDS-PAGE performed under both reduced and non-reduced conditions helps to further characterize the protein. Under non-reduced condition, it showed one band corresponding to high molecular weight protein of approximately 190 kDa (lanes 1 & 2) which seem to be the trimeric forms of a 50-55 kDa monomer as shown in lane 3 of (Fig. 4B). The corresponding α and β polypeptides were further confirmed by applying the BnC under reduced conditions. Addition of β-mercaptoethanol reduced the 50 kDa monomer releasing acidic and basic chains which are revealing the typical composition of this protein. This separated monomer in two smaller subunit ranges of approximately 28-32 (α) and 17-20 (β) kDa bands as shown in lanes 4 and 5 of (Fig. 4B). This confirms at least one inter-chain disulfide bond between α and β polypeptides which is very much typical for Cruciferins (Dalgalarrondo et al., 1986; Robin et al., 1991). Our results are in good agreement with already reported molecular weights of Cruciferin proteins under both reduced and non-reduced conditions. It has been shown that under reducing conditions, Cruciferin protein produced two major bands
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Chapter 4 Discussion which correspond to basic (MW 22-24 kDa) and acidic (MW 30-37 kDa) polypeptides linked by a disulfide bond (Dalgalarrondo et al., 1986; Robin et al., 1991). Cruciferin is composed of α- and β-polypeptides linked through a disulfide bond (Schwenke et al., 1983). Similarly, under non-reduced conditions, the molecular weight of the monomer is around 50 kDa and is made of 20-25 kDa acidic (β) chain and 30 kDa basic (α) chain linked by disulfide bonds (DeLisle & Crouch, 1989). Both of the chains are part of the same precursor single polypeptide molecule and are co-translationally synthesized in cytoplasm by rough endoplasmic reticulum or protein storage vacuoles. As already reported, the 50 kDa monomer oligomerizes by the help of hydrogen bonding (Bérot et al., 2005) and this has been seen in one dimensional SDS-PAGE. The electrophoresis was performed for a longer period of time to bring these oligomeric forms into the gel and therefore 50 kDa molecules had already left the gel (Fig. 4B; lanes 1 & 2). It is also possible that even larger oligomeric forms are detectable.
A primary sequence of 17 amino acids (GLEETICSMRSNEXEDD) from N- terminus was obtained through Edman degradation. Analysis of this sequence via protein BLAST (UniProtKB) further confirmed the protein as 12S globulin and showed a very high identity with other known Cruciferins of the Brassicaceae family as shown in (Table 3). The sequence showed 82% amino acid homology to that of 12S CRC Cruciferin from Arabidopsis thaliana (Hou et al., 2005). Further homology comparison suggested a high similarity to already known seed storage Cruciferins of the family Brassicaceae. It also showed high homologies with already reported Cruciferins from Raphanus sativus (Depigny et al., 1992), Brassica napus (Rödin et al., 1992) and to 11S globulins from Sinapsis alba (Palomares et al., 2005). Occurrence of Cruciferin in seeds of many different species is a good indication of their wide spectrum of occurrence among members of family Brassicaceae.
2D gel electrophoresis proved a useful technique towards identification of hetero- oligomeric subunit pairs and towards establishing relationship with their constituent polypeptide chains in seed proteins. The presence of a mother band with molecular weight of approximately 190 kDa proposed the presence of multiple isoforms hiding under the bands of 30 (α polypeptides) and 20 (β polypeptides) kDa ranges. To further analyze these different isoforms, two-dimensional electrophoresis (IEF/SDS-PAGE) was performed in order to separate these isoforms based on the differences between their
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Chapter 4 Discussion isoelectric points. As shown in (Fig. 5), α-polypeptide chains are separated in a molecular weight range between 28 to 32 kDa with pI ranges between 5.7 to 8.0 (red box). Similarly, the β-polypeptide is separated in a molecular weight range between 17 to 20 kDa with a pI range of 5.5 to 8.5 (yellow box). The separation of α- and β-polypeptide in pI ranges 6.7-8.8 and 5.9-9.5 respectively have already been reported (Nietzel et al., 2013). The molecular weights of rapeseed Cruciferins have been reported in the range of 240-350 kDa with pI of 7.2 (Schwenke,1994; Schwenke et al., 1981) in parallel to 300 kDa (Dalgalarrondo et al., 1986) for B. napus (L.). The two results are showing relatively high pH values of the acidic polypeptide as compared to already reported relatively lower values (Shewry et al., 1995). The little variation between the two results could be attributed either due to two different species or the difference of the extraction procedures.
Circular Dichroism (CD) data suggested the presence of both α-helical and β- sheeted secondary structure of the 190 kDa native Cruciferin, showing a broad negative ellipticity peak which is typical for α and β type protein (Manavalan et al., 1983) as shown in (Fig. 6A). The secondary structure content was calculated to approximately 30% α-helical structure, 15% turns and loops while major part (55%) is comprised of β- sheets. A comparative topology diagram of B. napus 11S globulins (PDB ID: 3KGL) illustrating the dominating anti-parallel β-strands is also shown in (Fig. 6B). The molecular structure of 11S seed globulin from B. napus has been determined and is deposited in Protein Data Bank via code 3KGL (Tandang-Silvas et al., 2010). The pro- 11S globulin has been expressed in E. coli and was then crystallized to solve the structure through x-ray crystallography. A brief analysis of the structure is showing the dominance of anti-parallel β-sheeted secondary structure over the helices which are just a minor part of the molecule as shown in (Fig. 6B). Similar properties have been observed in the CD spectra of B. nigra Cruciferin as the characteristic negative band at 222 nm from α-helices was not observed, in accordance with a lower content of α-helical structure of Cruciferin (Fig. 6A). Similar results and studies about the interaction between lomefloxacin (LMF) and human lactoferrin (Hlf) was studied by using fluorescence, circular dichroism (CD) spectroscopic and molecular modeling measurements.
Initially, the Cruciferin was extracted and purified in 50 mM phosphate buffer (pH 6.5), however, DLS showed a Polydispers signal as shown in (Fig. 7A) indicating the
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Chapter 4 Discussion presence of some high molecular weight oligomers at pH 6.5. Therefore, different buffers were tested through dialysis and ultimately the purified Cruciferin solution was adjusted to a concentration of 12 mg/ml in 20 mM Tris-HCl, pH 7.5. The solution was centrifuged, filtered using micro-spin filter tubes and subjected again to DLS measurements. A single prominent signal corresponds to a monodisperse protein solution (12 mg/ml) with RH of 5.8 ± 0.1 nm as shown in (Fig. 7B and 7C) along with the effect of the buffer on the removal of larger oligomers. A combined effect of pH and salt has been observed to influence the oligomerization of Cruciferins along with other physical factors. Such physical factors are influencing the intra and inter-chain hydrogen bonding of the surface occupied aliphatic side chains. A low ionic strength and pH near to physiological value favors the development of trimer development. Similar effects have already been observed on dissociation and association of mustard 12S proteins under different concentrations of SDS by using a variety of analytical techniques e.g., ultraviolet difference spectroscopy, gel filtration, fluorescence spectroscopy and viscometry (Rao & Rao, 1979). Under denaturing conditions, the larger complexes were dissociated in nearly ~15 bands of 60-12 kDa smaller sub-units. Nevertheless, antibodies directed immunoblots showed strong signals in the ranges of 30 and 20 kDa. Similarly, the rapeseed Cruciferins form hexameric assemblies with ionic strength above 0.5 M and the structure is breaking down to trimeric forms with lower values (Schwenke et al., 1983). Their results showed much variation in the physiochemical properties of the Cruciferin proteins and their respective complexes. Therefore, a more general correlation between Cruciferin oligomerization and physical parameters is difficult to conclude.
Assuming a globular shape, the corresponding molecular weight estimation is confirming the trimeric state of the native Cruciferin. Furthermore, native Cruciferin of B. nigra has a molecular mass of ~190 kDa, as determined by non-reduced SDS-PAGE (Fig. 4B; Lanes 1 & 2). DLS measurements verified the monodispersity of the solution and also indicated the trimeric nature of the protein. In 20 mM Tris buffer of pH 7.5, the major band of the trimer corresponding to approximately 190 kDa was found as indicated by the hydrodynamic radius of 5.8 nm (Fig. 7B). However, a much compact picture of the oligomeric forms of these Cruciferins has been reported from seeds of B. napus (Nietzel et al., 2013). They have analyzed a barrel-like octamer corresponding to a molecular weight of approximately 300-390 kDa in blue native PAGE. Such variation of oligomer size between Cruciferin proteins of different plant species could be due to their
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Chapter 4 Discussion extractions at different developmental stages of the seed in parallel to different in vitro physical parameters as already discussed before.
Flower like crystals (approximately 0.1 ×0.09×0.06 mm) appeared after nine months in multiple conditions of Morpheus screen as shown in (Fig. 8). Unfortunately these crystals diffract up to maximum 8 Å resolutions, which was unsuitable for structure determination and therefore, the data collection was stopped after processing of few images. The structural details of these storage proteins are of much interest to understand patterns of their molecular oligomerization and packaging inside the protein vacuoles as well as their functions at the molecular level. The trimeric state of Cruciferin in 20 mM
Tris-HCl, pH 7 was analyzed using SAXS (Fig. 9 and 10). The radius of gyration RG and the maximum dimension Dmax obtained from the collected data were 4.24 ± 0.25 nm and 14.0 ± 0.4 nm, respectively (Fig. 9A and 9B); these values suggest a folded but extended structure. Based on the shape of the Kratky plot curve, the protein is rather rigid and compactly folded (Fig. 9C). A conservation of the trimerization and a globular slightly oblate shape are strongly indicated as further deduced from the p(r) function (Fig. 9C).
The shape factor, RG divided by RH as determined by dynamic light scattering, has a value of 0.71 which is indicative for a nearly globular particle in agreement with the displayed ab initio model. Besides, calculated ab initio models are structurally highly similar compared to each other with an NSD value of 1.029 ± 0.112. In addition to these SAXS parameters, all other important values have been summarized in Table 2.
The scattering amplitude of Cruciferin processed by PRIMUSQT was compared to an 11S globulin from B. napus (PDB ID: 3KGL) by the program CRYSOL as shown in (Fig. 9A). The calculated minimized χ2-value of 1.337 for the comparison of the scattering pattern of Cruciferin from B. nigra (blue) and 11S globulin from B. napus (red) over a wide range of angles confirmed high structural similarity. A comparable molecular weight is suggested by the forward scattering intensity. Even the intensities at moderately higher scattering angles (s > 0.8 nm-1) are matching well pointing at the conservation of some structural detail. Similarly, the manual superimposition well confirms the conclusion that the structures are widely similar, including the molecular weight comparison based on the volume of the ab initio model. Specifically, a highly similar molecular weight and globular shape including a conserved trimerization of B. nigra Cruciferin is indicated. N-terminal sequence of B. nigra Cruciferin (β-polypeptide)
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Chapter 4 Discussion showed high identity (65%) with 3KGL indicating two very homologous primary sequences. 3KGL possesses a handsome number of positively and negatively charged amino acids (94) which are evenly distributed on the surface and are playing a significant role in the development of compact globular shape due to their electrostatic forces and we believe that same would be the case regarding the molecular shape of B. nigra Cruciferin. In compliance to this, the SAXS calculated a gyration radius (4.24 nm) confirming the globular shape of B. nigra Cruciferin protein. The globule state has been proposed as a major intermediate of protein folding.
The displayed ab initio dummy model with P3 symmetry was superimposed with the 11S globulins from B. napus (PDB ID: 3KGL) as prepared by PyMOL (Fig. 10). The 11S procruciferin protomer of B. napus (3KGL) is composed of three monomers which are joined to each other through non-covalently linked head to tail fashion and each monomer further exhibited a much conserved two jelly-roll β-barrel and two extended helix domains. The number and length of these α-helices and 310 helices is more varied than the β-stands and are making the extended loop region of the N and C terminal domains of these 11S globulins. Similarly, some of the proteins have short helices while in others, these helices are noticeably longer than others (Tandang-Silvas et al., 2010). The tentative ab initio model of Cruciferin (Fig. 10, grey spheres) displays an extended ―loop structure‖ sticking out of the nearly globular surface in agreement with the p(r) function. This feature may correspond to an extended terminus or loop of Cruciferin that possesses some flexibility in solution and thus is unique to B. nigra Cruciferin or might be not obvious in the crystal structure of the B. napus homologue. We believe that these extended flexible loops (indicated by black arrows in (Fig. 10) in addition to the micro- heterogeneity of indicated isoforms are responsible for the low resolution diffraction of the Cruciferin crystals. The location of these loops on the surface of the molecule is imparting the flexibility to the molecule and less organized packing inside the crystal which is responsible for the high mosaicity and ultimately the poor quality of diffraction data. Few of the solved molecular structures of different seed storage proteins from different plant species have already been reported. However, in all such structures, the heterogeneity of the native isoforms has been removed by cloning either the single gene or through the development of mutant cultivars which resulted in the production of high resolution diffracting crystals.
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In summary, we assume that seed storage proteins of B. nigra have similar properties perhaps forming a barrel-shaped hexamer. However, in buffers of lower ionic strength and different pH value, it is dissociating into a trimeric form of 190 kDa as approximated by DLS and SAXS measurements. Nevertheless, it is needed to solve the structure of native Cruciferins protein isolated and purified directly from plant, in order to have more detailed insights as well as to understand the oligomerization of these proteins.
Conclusions
The study includes the identification, purification and characterization of a trimeric seed storage Cruciferin (12S globulin) protein of Brassica nigra (L.). N-terminal amino acid sequence; obtained through Edman degradation, showed more than 80 % homology with already reported Cruciferin of Arabidopsis thaliana. One dimensional SDS-PAGE exhibited multiple protein band pattern of 50-55 kDa monomeric Cruciferin splitting into α-polypeptides of 28-32 kDa and β-polypeptides of 17-20 kDa under reduced conditions. The two dimensional electrophoretic analysis showed the further separation of the monomer into multiple isoforms with pI ranges between 5.7-8.0 (α-polypeptides) and 5.5-8.5 (β-polypeptides). The circular dichroism (CD) spectra revealed a calculated secondary structure content of 30% α-helix, 55% β-sheet and 15% turns. Dynamic Light Scattering (DLS) showed a hydrodynamic radius of 5.8 ± 0.1 nm confirming the trimeric nature of the protein. Cruciferin from Brassica nigra was crystallized and diffracted at 8 Å resolutions in 10 % PEG 4,000, 20 % (v/v) glycerol and 0.1 M trizma base (pH 8.5) and 0.02 M of each amino acid condition. Experimental SAXS analysis showed that the molecular shape of Cruciferin is globular and oligomeric state of the protein revealed a gyration radius of 4.24 ± 0.25 nm.
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4.2 Eruca sativa Cruciferin (EsC) A 50 E. sativa Cruciferin (EsC) has been isolated and analyzed by LC-MS/MS mass spectrometry. Seven peptides were identified from the 50 kDa protein which showed 63% sequence identity with Cruciferin (CRU1) protein from Raphanus sativus and CRU3 from Brassica napus as shown in (Fig. 11). The 12S Cruciferin with its polypeptide chains and the corresponding isoforms were analyzed by SDS- PAGE. Reduced SDS-PAGE showed the presence of two molecular weight ranges i.e., 30 kDa and 20 kDa when extracted and purified in phosphate buffer at pH 7.0 as shown in (Fig. 12B, Lane 2). These results are in good agreement with already reported molecular weights of Cruciferin proteins under both reduced and non-reduced conditions. It has been shown that under reducing conditions, Cruciferin protein produced two major bands which correspond to basic (MW 22-24 kDa) and acidic (MW 30-37 kDa) polypeptides linked by a disulfide bond (Dalgalarrondo et al., 1986; Robin et al., 1991).
The molecular structure of 11S seed globulin from B. napus has been determined and is deposited in Protein Data Bank via code 3KGL (Tandang-Silvas et al., 2010). The pro-11S globulin has been expressed in E. coli and was then crystallized to solve the structure through x-ray crystallography. A brief analysis of the structure is showing the dominance of anti-parallel β-sheeted secondary structure over the helices which are just a minor part of the molecule as shown in (Fig. 13). Similar properties have been observed in the already described CD spectra of BnC as the characteristic negative band at 210 nm for α-helices was not observed which is in accordance with a lower content of α-helical conformation of EsC (Fig.13).Secondary structure of EsC showed a well folded protein and CD spectrum results indicted 7.5% α-helix, 47.6% β-sheet, 7 % turn, 37.9 % not compactly folded (random), along with an RMS value of 6.9 for the respective reference fit curve.
Furthermore, DLS measurements verified the monodispersity of the solution and also indicated the trimeric nature of EsC with hydrodynamic radius of 5.5 nm (Fig. 14A and 14B) and this trimeric nature was further verified by gyration radius (4.3 nm) after analyzing SAXS data. Triangle like crystals of approx. (0.1 × 0.05 × 0.05 mm) sizes were appeared after 10 days in E11 of Morpheus screen condition as shown in (Fig. 15). Unfortunately these crystals diffracted up to maximum 7 Å resolutions, which was
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Chapter 4 Discussion unsuitable for structure determination and therefore, the data collection was stopped after processing of few images.
The structural details of EsC are of much interest to understand patterns of their molecular oligomerization and packaging inside the protein vacuoles as well as their functions at the molecular level. The trimeric state of EsC in 20 mM Tris-HCl, pH 7.5 was analyzed using SAXS (Fig. 16 and 17). The radius of gyration Rg and the maximum dimension Dmax obtained from the collected data were 4.3± 0.30 nm and 15.0 ± 0.41 nm, respectively (Fig. 16 A and B), these values suggest a folded but extended structure. Based on the shape of the Kratky plot curve, the protein is rather rigid and compactly folded (Fig. 16 B). A conservation of the trimerization and a globular slightly oblate shape are strongly indicated as further deduced from the p(r) function (Fig. 16 C). The shape factor, Rg divided by RH as determined by dynamic light scattering, has a value of 0.76 which is indicative for a nearly globular particle in agreement with the displayed ab initio model. Besides, calculated ab initio models are structurally highly similar compared to each other with an NSD value of 0.957 ± 0.271.
The scattering amplitude of EsC processed by PRIMUSQT was compared to an 11S globulin from B. napus (PDB ID: 3KGL) by the program CRYSOL as shown in (Fig. 16A). The calculated minimized χ2-value of 2.09 for the comparison of the scattering pattern of EsC from E. sativa (green) and 11S globulin from B. napus (blue) over a wide range of angles confirmed high structural similarity. A comparable molecular weight is suggested by the forward scattering intensity. Similarly, the manual superimposition well confirms the conclusion that the structures are widely similar, including the molecular weight comparison based on the volume of the ab initio model.
The displayed ab initio dummy model of EsC with P3 symmetry was superimposed with the 11S globulins from B. napus (PDB ID: 3KGL) as prepared by PyMOL (Fig. 17). The 11S procruciferin protomer of B. napus (3KGL) is made up of three monomers which are joined to each other through non-covalently linked head to tail fashion and each monomer further exhibited a much conserved two jelly-roll β-barrel and two extended helix domains. The number and length of these α-helices and 310 helices is more varied than the β-stands and are making the extended loop region of the N and C terminal domains of these 11S globulins. Similarly, some of the proteins have short helices while in others, these helices are noticeably longer than others The tentative ab Page 96
Chapter 4 Discussion initio model of EsC (Fig. 17, grey spheres) displays an extended ―loop structure‖ sticking out of the nearly globular surface in agreement with the p(r) function. This feature may correspond to an extended terminus or loop of Cruciferin that possesses some flexibility in solution and thus is unique to EsC or might be not obvious in the crystal structure of the B. napus homologue. We believe that these extended flexible loops (indicated by black arrows in Fig. 17) in addition to the micro-heterogeneity of indicated isoforms are responsible for the low resolution diffraction of the EsC crystals. The location of these loops on the surface of the molecule is imparting the flexibility to the molecule and less organized packing inside the crystal which is responsible for the high mosaicity and ultimately the poor quality of diffraction data. Few of the solved molecular structures of different seed storage proteins from different plant species have already been reported. However, in all such structures, the heterogeneity of the native isoforms has been removed by cloning either the single gene or through the development of mutant cultivars which resulted in the production of high resolution diffracting crystals.
Conclusions
The study describes the identification, purification and characterization of a trimeric seed storage Cruciferin (12S globulin) protein of Eruca sativa (L.). LC-MS/MS mass spectrometric analysis showed 63% sequence identity with Cruciferins (CRU1) of Raphanus sativus and Brassica napus (CRU3) One dimensional SDS-PAGE exhibited multiple protein band pattern of 50-52 kDa monomeric Cruciferin splitting into α-polypeptides of 30 kDa and β-polypeptides of 20 kDa under reduced conditions. The circular dichroism (CD) spectra revealed a calculated secondary structure content of 7.5 % α-helix, 47.6 % β-sheet, 7 % β-turn and 37.9 %. Dynamic Light Scattering (DLS) showed a hydrodynamic radius of 5.5 ± 0.3 nm confirming the trimeric nature of the protein. EsC from Eruca sativa was crystallized and diffracted at 7 Å resolutions. Experimental SAXS analysis showed that the molecular shape of EsC is globular and oligomeric state of the protein revealed a gyration radius of 4.3 ± 0.30 nm.
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4.3 Sisymbrium irio Ubiquitin (SiU)
Here we describe the identification, purification and secondary structure of an Ubiquitin protein from seeds of Sisymbrium irio. Isolated S. irio Ubiquitin (SiU) has a molecular mass of around 17 kDa as visualized on SDS-PAGE (Fig. 18C, lane L1). Ubiquitins in plants exist either as mono Ubiquitins or poly Ubiquitins (Komander et al., 2009). Most of the Ubiquitins have molecular masses in the range of 8.5 kDa and show similarities in their amino acid sequences. Isoelectric pH (pI) of isolated Ubiquitin by isoelectric focusing experiment was found as 9.2 which are very near to the theoretical pI of reported Ubiquitins. The amino acid sequence of SiU was obtained by LC–MS/MS which provided five peptide fragments (Table 6). Sequence similarity search in UniProtKB for individual peptides provided more than 88% sequence identity with Arabidopsis thaliana poly Ubiquitin (Uni prot ID: Q39256.1) and Caenorhabditis elegans (Uniprot ID: P0CG71.1). The combined sequence showed 77 % identity with Ubiquitins from Triticum aestivum (Uniprot ID: P69326.2) and Avena sativa (Uniprot ID: P69310.1) (Table 7).
CD spectroscopy data of SiU indicated the presence of both α-helix and β-sheet secondary structural elements in the native fold but mostly it comprised of β-sheet and random coils. The secondary structure estimation for the whole protein SiU by CD spectroscopy and the secondary structure prediction by Quick 2D for the obtained sequence of SiU matches well with each other. It also indicates secondary structure conservation within the reported Ubiquitins with more content of β-sheet structure over α- helix. The obtained SiU sequence was submitted to Quick 2D for the prediction of secondary structural elements which showed β-sheet conformation for amino acid sequence 2TITLEV7, 20LIF22 and 43TLHLVLR49. However, the peptide fragment 12TIDNVKAK19 formed α-helix structure. Secondary structural elements of SiU revealed that β2 contains residues 12-17, α1 expresses 22-28 residues, β3 holds 43-46 amino acids, β4 comprises 48-49 residues, η2 encloses 56-60 amino acids, β5 constitutes 63-69 amino acids. Different structural motifs like F4 patch, TEK box, I36 patch and I44 patch are also available and conserved in SiU (Fig. 23B).
DLS showed hydrodynamic radius 2.44 with 17 kDa size of SiU protein was compared with the standard hydrodynamic radius of globular proteins (Strobl, 1997), which might infers the existence of an open conformation of either M1 or K63-linked di
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Ubiquitin dimer in solution. Structural characterization of different di Ubiquitin molecules revealed that different linkages between monomers result in distinct chain conformations; i.e., compact and open. In the prevalent model for K48-linked Ubiquitin, the monomer chains interact via their I44 patches, and two such Ubiquitin modules pack tightly in tetra Ubiquitin molecule (Cook et al., 1992; Varadan et al., 2002; Tenno et al., 2004; Eddins et al., 2007). These types of association through K48-linked chains mostly adopt compact conformations. Similar to K48 linkages, K6- and K11-linked di Ubiquitin adopts compact conformations (Bremm et al., 2010; Matsumoto et al., 2010; Virdee et al., 2010) while M1 and K63-linked di Ubiquitin mostly exhibits open conformations (Varadan et al., 2004; Datta et al., 2009; Komander et al., 2009; Weeks et al., 2009).
In order to analyze the conserved features of the Ubiquitin sequence, a multiple sequence alignment of the combined peptide sequence of S. irio sequence and other homologous Ubiquitins is performed (Fig. 21). Multiple sequence alignment suggested that Ubiquitin proteins contain overall conserved sequences with very few non-conserved amino acids. SiU sequence is also found well conserved among different Ubiquitins with conserved changes of A28 and A57 while non-conserved changes K42, D49, and D62. A homology model of the obtained sequence of SiU was constructed and compared with human Ubiquitin for different structural features. Due to incomplete sequence, the modeled SiU has missing β1 strand, lower part of α-helix (α1) and complete 3-10 helix (η1) while other structural features are well conserved. The presence of seven lysines is the most unique feature of Ubiquitins as already have been described and these lysines are very actively involved in a variety of their functions. In compliance to this, it is very interesting to note the mutation at position 42 where arginine has been replaced with another lysine, so altogether there has been the presence of eight lysines in the fragmented primary sequence of SiU. The presence of extra lysine has really made the things interesting to know its role in the interaction of SiU with other molecules. The presence of more lysine in gap areas of SiU primary sequence may also be possible. Similarly, the replacement of two glutamines with negatively charged aspartates is also point of consideration as it might result in conformational change of SiU and ultimately the corresponding function. These unique and typical changes really are tempting towards the detailed analysis of SiU molecular structure and also the interaction mechanisms with other molecules. These mutations may also be taken as an evidence of variation among very conserve Ubiquitin sequence and quite possible that the discovery of more
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Ubiquitins from different organisms lead towards the sequence variety and ultimate function diversity of these important proteins. Hence, the crystallization attempts of this protein are underway to solve the native three dimensional structure of this important protein.
Conclusions
A 17 kDa Ubiquitin was identified, characterized and purified by LC–MS/MS and size-exclusion chromatography from the seeds of Sisymbrium irio. It has around 77% sequence identity with Ubiquitin proteins from Triticum aestivum, Avena sativa, Arabidopsis thaliana and others. Secondary structure of SiU consisted of about 6.5% α-helix, 39.3% β-sheet, 0.7 % turn, 53.5 % random coil which indicates that it is rich in β-sheet. Result of Multiple sequence alignment of 20 different Ubiquitin sequences showed that Ubiquitin sequence is well conserved in nature with very few and unique amino acids mutations (R42 by K42, Q49 and Q62 by D49 and D62, respectively). Homology modeling of SiU (11-28, and 42-76) residues suggested the overall conserved secondary and tertiary structure and other structural motifs.
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4.4 Brassica nigra Napin (BnN)
Napins (2S albumin) are seed storage proteins (SSP) which are used by plant as a source of carbon and nitrogen and also play key role in plant defense. An approximately 16 kDa Napin protein has been identified and purified from seeds of Brassica nigra. However, seed Napins with varying molecular weights have also been reported from different plants of Brassicaceae and other plants families (Venkatesh &Rao, 1988). It belongs to super family prolamins (Shewry et al., 1995). Napin is the second abundant protein in Brassicaceae after Cruciferin; ranging from 15 to 45% of SSP depending on the variety and species.
The identification of BnN was done by feeding N-terminus amino acid sequence in UniProtKB online server which provided 78.8% sequence identity to already reported
Napin (2S albumin) of Raphanus sativus and 71.4% to that of Brassica campestris (Table 8). Further identification was confirmed by analyzing the purified BnN on reduced SDS- PAGE where it dissociated in two polypeptides of approximately 10 and 4 kDa as shown in lane 2 of (Fig. 24B). However, the low molecular weight band of 4 kDa was not appearing on the gel. These results indicated that BnN is composed of two polypeptide chains and these chains are linked with each other by disulfide bond which is very much typical to Napin family of proteins. The location of cysteine and number of cysteine residues in the polypeptide chain are conserved features of Napin and are already well documented. Eight cysteine residues (8 Cys motif), two in the short chain and six in the long chain have been identified from different Napin isoforms (D'Hondt et al., 1993). Disulfide bonds play an important role in three-dimensional molecular structure determination of Napin. This feature is leading to a compact structure that is difficult to access by digestive enzymes (Gehrig & Biemann, 1995). Similarly, the Napins from B. juncea have molecular weight ranging from 12 to 13 kDa and are composed of two polypeptides of 9 and 4 kDa (Venkatesh & Rao, 1988). S. alba Napin possesses molecular weight of 14.6 kDa and is composed of two polypeptide chains of 9.5 and 5 kDa (Menéndez et al., 1987).
The secondary conformation of BnN was analyzed by circular dichroism (CD) spectroscopy in far UV (190–260 nm) range. BnN was dissolved in 25mM phosphate buffer (pH 7) and CD spectrum was obtained at 25 °C as shown in (Fig. 25). The spectra
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Chapter 4 Discussion showed two minima at 210 and 225 nm which indicated a high α-helical content (38%) and relatively lower β-sheeted (20%) conformation. Interestingly such a high content of α -helix has already been reported with other Brassicaceae Napins e.g., Napin of S. alba have 50% α-helix and 5% β-sheets in the molecule (Menendez et al., 1987).
Further molecular characterization of BnN was done by measuring its hydrodynamic radius by Dynamic Light Scattering (DLS) instrument. DLS calculated RH value of 1.99 nm which very much coincides with 16 kDa molecular weight of Napin protein as per the standard hydrodynamic radii calculations of globular proteins (Strobl, 1997) and it further infers the existence of a monomer in the solution. The BnN was concentrated up to 15 mg/ml and exposed to 4 and 20 °C temperatures in order to optimize the crystallization conditions. Monodispersity and stability of BnN was observed for three consecutive days at 4 and 20 °C by DLS. Effect of low and high temperature treatment on stability has been shown in (Fig. 27 and 28). After ensuring the stability of concentrated (15 mg/ml) BnN, it was put to more than 500 crystallization conditions and diamond shaped microcrystals appeared which unfortunately diffracted to poor resolution. Multigene families are involved in the expression of Napin in Brassicaceae plants e.g., the B. napus identified genes are napA, napB, BngNAP1, gNa and three cDNA sequences pN1, pN2 and pNAP1 (later identified as napA) (Gehrig &Biemann, 1995) out of 10-16 multigene. This multiple gene expression might result in the production of closely related BnN isoforms and this produced crystals of high mosaicity which causes the diffraction data to poor resolutions (Fig. 29).
Many plants released toxic metabolites with antimicrobial activity and are currently commercially available as they also show some toxicity towards insect pests. The antifungal activity of BnN was tested against phytopathogenic Fusarium solani and Fusarium oxysporum fungi. Purified BnN (30 µg) inhibited the growth of these two fungi as shown in (Fig. 30A and 30B). Terras et al. (1993) have already reported the antifungal activity of rapeseed Napin (2S albumins) against different fungi. Recently, Lin & Ng (2008) has reported that a Napins-like polypeptide often shows antifungal activity with high thermo stability. Two structural requirements have been shown to be necessary for antifungal activity of these Napins (Powers & Hancock, 2003), first an amphipathic Napin conformation and secondly a cationic charge distribution e.g., Napins of Rapeseed showed strong amphipathic behavior parallel to their high lysine and arginine content,
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Chapter 4 Discussion and thus fully comply with these two requirements. Amphipathic α-helix structure of Napins may be involved in the CaM (Calmodulin) antagonist and formation of pores in membranes (Neumann et al., 1996b). This is probably because CaM and two subunits of Napins contain similar α-helical conformations. Another assumption concerning the antifungal mechanism of Napins may be due their role towards protease inhibition which leads to an alteration of protein metabolism (Neumann et al., 1996a).
Due to the alarming situation regarding the toxic impacts of conventional chemical insecticides on human health and surroundings, the search for novel molecules with insecticidal activity with minimal adverse effects has become paramount (Da Silva et al., 2012). Consequently, entomo-toxic plant compounds are an appreciated approach to develop bio-insecticides against stored product insect pests (Bednarek & Osbourn, 2009) on a long- term scale after carefully verifying their respective persistency and toxicity spectrum. Many promising biochemical molecules have been produced in the plants that act as the defense against the attack of pest on plants, such as small cysteine- rich peptides for establishment of constitutive resistance against insect pests (Silverstein et al., 2005). Purified Napin especially of the 3 mg/ml concentration significantly reduced the three life stages (i.e., larval, pupal and insecticidal) of T. castaneum population in comparison to control and thus proves to be a promising candidate.
Conclusions
A 16 kDa Napin was identified by N-terminal amino acid sequence and further purified by a combination of Cation exchange and size-exclusion chromatography from seeds of Brassica nigra. CD spectrum showed that BnN consists of about 38 % α-helix, 20 % β-sheet, 11 % turn. DLS data indicated the monodispersity and Stability of BnN which ultimately crystallized to diamond shaped crystals but diffracted to poor resolution. Functional characterization of BnN was performed by doing the activity assays against fungi and insects. BnN significantly inhibited the growth of both Fusarium solanii and Fusarium oxysporum as well as significantly inhibited the T. castaneum growth.
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4.5 Precursor Eruca sativa Thionin (pEsT)
Thionins are present in leaves, seeds, roots and stems of a number of plant species e.g., mistletoes, cereals and crucifers. Thionins are synthesized as larger precursors, which have a post-translational N-terminal signal peptide and a C-terminal precursor peptide. Thionins from the seeds of Taramira (Eruca sativa), a member of the family Brassicaceae, has been isolated and characterized. Different types of Thionins from different plants, namely viscotoxins (Pal et al., 2008), α-hordothionin (Johnson et al., 2005), α-purothionin (Teeter et al., 1990) and Thionins 2.4 (Asano et al. 2013) have already been reported. The identification of pEsT was done by feeding the MALDI- TOF/TOF mass spectrometric residual data in UniProtKB online server which provided
100 % sequence identity to already reported Thionin (Thi 2.4) of Arabidopsis thaliana (Fig. 31). Further identification was confirmed by analyzing the purified pEsT on SDS- PAGE. Precursor pEsT has a molecular mass of around 16 kDa including the signal peptide, Thionin and acidic part together, however in the presence of some reducing agent like Dithiothreitol or β-mercaptoethanol, this native molecule is splitting in two smaller subunits of 11 kDa (acidic domain) and 5 kDa (Mature Thionin) as determined by SDS- PAGE as shown in Fig.33 A and 33B (lane 1 & 2).
Precursor EsT (pEsT) circular dichroism (CD) spectra were analyzed according to Yang (Yang et al., 1986) as shown in Fig. 34, which indicated that the precursor Thionin is rich in α-helix (38 %) with a convincingly low RMS value of 2.995. Mature Thionin has also been reported to possess the α-helical dominating conformation and no information is present about the isolated acidic domain. However, our CD spectrum is also indicating a high α-helical content also in the acidic domain as well. The high content of α-helix in pEsT is considered to be responsible for the toxic biological activities of this protein towards plant pathogens and also facilitates the dynamic association of the pEsT with membranes; thus resulting in the increase membrane permeability of the pathogen which leads towards their death (Bechinger, 2010).
Further molecular characterization of pEsT was done by measuring its hydrodynamic radius by Dynamic Light Scattering (DLS) instrument as per the standard hydrodynamic radii calculations of globular proteins (Strobl, 1997). DLS calculated RH value of 2.5 nm which is indicating a relatively higher molecular weight of around 25
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Chapter 4 Discussion kDa for the precursor molecule, however, since the native molecule is not that globular in shape, so its bit misleading while the correct molecular weight is about 16 kDa as indicated by the SDS-PAGE and size exclusion chromatography and it further infers the existence of a monomer in the solution. The pEsT was concentrated up to 25 mg/ml and exposed to 4 and 20 °C temperatures in order to optimize the crystallization conditions. Monodispersity and stability of pEsT was observed for three consecutive days at 4 and 20 °C by DLS. Effect of low and high temperature treatment on stability has been shown in (Fig. 36 and 37) After ensuring the stability of concentrated (25 mg/ml) pEsT, it was put to more than 600 crystallization conditions and diamond shaped microcrystals appeared which unfortunately diffracted to poor resolution (Fig. 38). The reason of low diffraction is because of the presence of acidic domain in the precursor molecule which is rather more flexible which in turn is contributing poor resolution the crystals.
The structural details of pEsT were determined by Small Angle X-ray Scattering (SAXS). SAXS results indicated that pEsT in elongated structure rather than globular shape in native form and it exist in monomer form with P1 symmetry as shown in (Fig. 39
C). The shape factor, Rg divided by RH as determined by dynamic light scattering, has a value of 0.8 which is indicative for a nearly elongated shape of pEsT particle in agreement with the displayed ab initio model in (Fig. 39C). The volume of pEsT by the ab initio model is 31100 nm3, corresponding to approximately 15.5 kDa (Fig. 39C) and this was most equal to MW shown on SDS-PAGE in lane 1 (Fig. 33B). Besides, calculated ab initio models are structurally highly similar compared to each other with an NSD value of 1.633 ± 0.238 (Fig. 39C).
Several plant Thionins are cytotoxic and have shown anti-cancerous activities. The peptides of these proteins can be utilized for the design of new anti-cancerous drugs. pEsT showed the cytotoxic activity against the Huh7 cells and IC50 was obtained at ≥ 12.5 µM but less than 15.63 µM as shown in the (Fig. 40). Romagnoli et al. (2003) isolated and purified the viscotoxins c1 from Viscum album ssp. Coloratumohwi and its cytotoxicity was estimated by inhibition of 3H-thymidin incorporated into Y. Sarcoma cells. LD50 was calculated as 0.198 µM which shows the intermediate toxicity with respect to the other reported viscotoxins. Loeza et al. (2008) used to express the cDNA of Thi 2.1 Thionin from Arabidopsis thaliana to the BVE-E6E7 bovine endothelial cell line and found its antibacterial, antifungal and cytotoxic activity. The cytotoxic activity of the
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Chapter 4 Discussion conditioned medium of clone EC-Thi2.1 was assessed by different normal and transformed mammalian cell lines which showed different percentages of inhibition. Johansson et al. (2003) identified four Thionins (phoratoxins C–F) from Phoradendron tomentosum and studied their cytotoxicity against ten different cell lines. They examined the LC50 values of all four proteins in the range of 0.04 to 0.83 µM and found that phoratoxins C is the most potent cytotoxic. Phoratoxins C was tested on primary cultures of tumor cells from patients and had a selective activity to breast cancer cells from tumor samples. The cytotoxicity and anticancer activity of Thionins are allocated to a cellular response that plays a role in the activation of Ca+2 influx coupled ion and it is involve in depolarization of the plasma membrane. Endogenous phospholipase A2 become activate as a result of plasma membrane depolarization due to this, membrane being to start alteration, and finally the cell death.
Thionins proteins are a family of protein which are rich in arginine, lysine, and cysteine residues and have a strong antimicrobial activity (Charity et al., 2005). pEsT from Eruca sativa exhibited antifungal activity against Fusarium solani and Fusarium oxysporum at 30 µg quantity. Chandrashekhara et al. (2010) reported that Thionin (PR protein-13) from Pennisetum glaucum (pearl millet) inhibited the growth of Sclerospora graminicola spores at a quantity of 100 µg. Wheat purothionin showed antifungal activity against Rhizoctonia solani by membrane permeabilization which is responsible for significant crop losses and rice sheath blight (Oard et al., 2004). Furthermore, Giudici et al. (2004) reported very strong antifungal activity of viscotoxins A3 and B that inhibited spore germination and mycelial growth of three fungal species; the results have been summarized in (Table 11).
Purified pEsT protein reduced the population of T. castaneum significantly as a promising starting point. Many plant released toxic metabolites with antimicrobial activity are currently commercially available as they also show some toxicity towards insect pests. Consequently, entomo-toxic plant compounds are an appreciated approach to develop bio-insecticides against stored product insect pests (Bednarek & Osbourn, 2009), on a long-term scale after carefully verifying their respective persistency and toxicity spectrum (Stephen et al., 2014) characterized pea albumin PA1b and mapped its binding site on insect vacuolar ATPase. Its interaction is influencing the toxicity, and a similar mechanism is conceivable in case of Thionin resulting in reduced T. castaneum
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Chapter 4 Discussion populations, as also observed. Da Silva et al. (2012) used the saponin 3-GlcA-28- AraRhaxyl-medicagenate from Medicago truncatula seeds due to its high toxicity against Sitophilus oryzae. This 3-GlcA-28-AraRhaxyl-medicagenate exhibits repellent properties and has a CMC of about 0.6 mM. However, there could be many reasons for toxicity of pEsT Thionins against T. castaneum as described previously (Gressent et al., 2003; Jouvensal et al., 2003; Rahioui et al., 2007; Muench et al., 2014). Experiments applying Thionin of Pyrularia, which is hemolytic, cytotoxic and neurotoxic, suggest that negatively charged membrane lipids are targeted directly (Castro & Vernon, 1989). Consequently, it is concluded that Thionins commonly do not possess receptor specificity but result in the formation of oligomeric protein-lipid complexes and ion permeable membrane pores (Hughes et al., 2000; Stec et al., 2004) acting on a broad spectrum of species. Nonetheless, an improved understanding of the structure-function relationship and mode of action is essential for biotechnological and medical applications.
Conclusion
A 16 kDa pEsT was identified, and purified by MALDI-TOF/TOF mass spectrometry and size-exclusion chromatography from the seeds of Eruca sativa. pEsT 100 % and 32 % sequence identity with Thionin 2.4 from Arabidopsis thaliana and THN of Brassica rapa respectively. Secondary structure of pEsT was calculated by CD spectrum which consisted of about 38 % α-helix, 9 % β-sheet, 19 % turn, 34 % not compactly folded (random) content. Monodispersity and Stability of pEsT were checked by DLS data to continuous three days at two different temperatures (4 and 20 °C). Micro rectangular shape crystals of pEsT were diffracted at poor resolution An elongated shape of pEsT was determined by small angle x-ray scattering (SAXS) experiment.
pEsT showed the cytotoxity against the Huh7 cells and LC50 was obtained at ≥ 12.5 µM but less than 15.63 µM. Antifungal activity of 30 µg 50 µg and 100 µg of pEsT was checked against the growth of Fusarium graminearum by 96 well-microtiter plate while Disc Diffusion method was used against Fusarium solani and Fusarium oxysporum. pEsT inhibited the growth of these fungi significantly.
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Entomotoxic activity of pEsT protein was checked against T. castaneum growth which was significantly inhibited by the protein.
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REFERENCES
Chapter 5 References
Aguilar, R.C. and B. Wendland. 2003. Ubiquitin: not just for proteasomes anymore. Current Opinion in Cell Biology, 15: 184-190.
Al-Jaber, N.A. 2011. Phytochemical and biological studies of Sisymbrium irio L. Growing in Saudi Arabia. Journal of Saudi Chemical Society, 15: 345-350.
Altschul, S.F., W. Gish, W. Miller, E.W. Myers and D.J. Lipman. 1990. Basic local alignment search tool. Journal of Molecular Biology, 215: 403-410.
Altschul, S.F., T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller and D.J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25: 3389-3402.
Aluko, R.E., T. McIntosh and F. Katepa‐Mupondwa. 2005. Comparative study of the polypeptide profiles and functional properties of Sinapis alba and Brassica juncea seed meals and protein concentrates. Journal of the Science of Food and Agriculture, 85: 1931-1937.
Aluko, R.E., M. Reaney, T. McIntosh, F. Ouellet and F. Katepa-Mupondwa. 2004. Characterization of a calcium-soluble protein fraction from yellow mustard (Sinapis alba) seed meal with potential application as an additive to calcium-rich drinks. Journal of Agricultural and Food Chemistry, 52: 6030-6034.
Alva, V., S.Z. Nam, J. Söding and A.N. Lupas. 2016. The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis. Nucleic Acids Research, 44: W410-W415.
Amri, E. 2014. The role of selected plant families with dietary Ethnomedicinal species used as anticancer. Journal of Medicinal Plants, 2.
Arayno, M. and N. Zafor. 1983. Phenolic compounds of Sismbruym incisum. J. Pharm, 2.
Arnold, K., L. Bordoli, J. Kopp and T. Schwede. 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics, 22: 195-201.
Page 110
Chapter 5 References
Asano, T., A. Miwa, K. Maeda, M. Kimura and T. Nishiuchi. 2013. The secreted antifungal protein thionin 2.4 in Arabidopsis thaliana suppresses the toxicity of a fungal fruit body lectin from Fusarium graminearum. PLoS Pathog, 9: e1003581.
Ashton, F.M. 1976. Mobilization of storage proteins of seeds. Annual Review of Plant Physiology, 27: 95-117.
Awaad Amani, A., D. Maitland and G. Soliman. 2006. Antiulcerogenic Activity of Alhagi maurorum. Pharmaceutical Biology, 44: 292-296.
Bagavathiannan, M. and R. Van Acker. 2009. Transgenes and national boundaries–The need for international regulation. Environmental Biosafety Research, 8: 141-148.
Baum, J.A., T. Bogaert, W. Clinton, G.R. Heck, P. Feldmann, O. Ilagan, S. Johnson, G. Plaetinck, T. Munyikwa and M. Pleau. 2007. Control of coleopteran insect pests through RNA interference. Nature Biotechnology, 25: 1322-1326.
Bechinger, B., 2010. Membrane association and pore formation by alpha-helical peptides. Proteins Membrane Binding and Pore Formation, 24-30.
Bednarek, P. and A. Osbourn. 2009. Plant-microbe interactions: chemical diversity in plant defense. Science, 324: 746-748.
Bell, J. 1993. Factors affecting the nutritional value of canola meal: a review. Canadian Journal of Animal Science, 73: 689-697.
Bell, L. and C. Wagstaff. 2014. Glucosinolates, myrosinase hydrolysis products, and flavonols found in rocket (Eruca sativa and Diplotaxis tenuifolia). Journal of Agricultural and Food Chemistry, 62: 4481-4492.
Benkert, P., M. Biasini and T. Schwede. 2011. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics, 27: 343-350.
Bennett, R.N., E.A. Rosa, F.A. Mellon and P.A. Kroon. 2006. Ontogenic profiling of glucosinolates, flavonoids, and other secondary metabolites in Eruca sativa (salad rocket), Diplotaxis erucoides (wall rocket), Diplotaxis tenuifolia (wild rocket),
Page 111
Chapter 5 References
and Bunias orientalis (Turkish rocket). Journal of Agricultural and Food Chemistry, 54: 4005-4015.
Bero, J., M. Frédérich and J. Quetin‐Leclercq. 2009. Antimalarial compounds isolated from plants used in traditional medicine. Journal of Pharmacy and Pharmacology, 61: 1401-1433.
Bérot, S., J. Compoint, C. Larré, C. Malabat and J. Guéguen. 2005. Large scale purification of rapeseed proteins (Brassica napus L.). Journal of Chromatography B, 818: 35-42.
Bhatty, R. and A. Finlayson. 1973. Extraction of nonprotein nitrogen from oilseed meals with different solvents. Cereal Chemistry, 50: 329-335.
Bhatty, R., S. McKenzie and A. Finlayson. 1968. The proteins of rapeseed (Brassica napus L.) soluble in salt solutions. Canadian journal of Biochemistry, 46: 1191- 1197.
Bhatty, R. and F. Sosulski. 1972. Diffusion extraction of rapeseed glucosinolates with ethanolic sodium hydroxide. Journal of the American Oil Chemists Society, 49: 346-350.
Biasini, M., S. Bienert, A. Waterhouse, K. Arnold, G. Studer, T. Schmidt, F. Kiefer, T.G. Cassarino, M. Bertoni and L. Bordoli. 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research: gku340.
Bilodeau, P., J.-G. Lafontaine and G. Bellemare. 1994. Far upstream activating promoter regions are responsible for expression of the BnC1 cruciferin gene from Brassica napus. Plant Cell Reports, 14: 125-130.
Björkman, M., I. Klingen, A.N. Birch, A.M. Bones, T.J. Bruce, T.J. Johansen, R. Meadow, J. Mølmann, R. Seljåsen and L.E. Smart. 2011. Phytochemicals of Brassicaceae in plant protection and human health–Influences of climate, environment and agronomic practice. Phytochemistry, 72: 538-556.
Page 112
Chapter 5 References
Blanchet, C.E., A. Spilotros, F. Schwemmer, M.A. Graewert, A. Kikhney, C.M. Jeffries, D. Franke, D. Mark, R. Zengerle and F. Cipriani. 2015. Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY). Journal of Applied Crystallography, 48: 431-443.
Bones, A., O. Thangstad, O. Haugen and T. Espevik. 1991. Fate of myrosin cells: characterization of monoclonal antibodies against myrosinase. Journal of Experimental Botany, 42: 1541-1550.
Bones, A.M. and J.T. Rossiter. 1996. The myrosinase‐glucosinolate system, its organisation and biochemistry. Physiologia Plantarum, 97: 194-208.
Bones, A.M. and J.T. Rossiter. 2006. The enzymic and chemically induced decomposition of glucosinolates. Phytochemistry, 67: 1053-1067.
Boydston, R. 2004. Take cover from the elements-Brassica cover crops can control weeds and reduce the use of crop protectants in vegetable rotations. American Vegetable Grower: 18-19.
Boyle, V.J., M.E. Fancher and R.W. Ross. 1973. Rapid, modified Kirby-Bauer susceptibility test with single, high-concentration antimicrobial disks. Antimicrobial Agents and Chemotherapy, 3: 418-424.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.
Bremm, A., S.M. Freund and D. Komander. 2010. Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nature Structural & Molecular Biology, 17: 939-947.
Chandrashekhara, S. Niranjan-Raj, S. Deepak, G. Manjunath and H. Shekar Shetty. 2010. Thionins (PR protein-13) mediate pearl millet downy mildew disease resistance. Archives of Phytopathology and Plant Protection, 43: 1356-1366.
Page 113
Chapter 5 References
Charity, J.A., P. Hughes, M.A. Anderson, D.J. Bittisnich, M. Whitecross and T. Higgins. 2005. Pest and disease protection conferred by expression of barley β- hordothionin and Nicotiana alata proteinase inhibitor genes in transgenic tobacco. Functional Plant Biology, 32: 35-44.
Collaborative, C.P. 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallographica. Section D, Biological Crystallography, 50: 760.
Cook, W., L. Jeffrey, M. Carson, Z. Chen and C. Pickart. 1992. Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2). Journal of Biological Chemistry, 267: 16467-16471.
Crouch, M.L. and I.M. Sussex. 1981. Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro. Planta, 153: 64-74.
D'Hondt, K., D. Bosch, J. Van Damme, M. Goethals, J. Vandekerckhove and E. Krebbers. 1993. An aspartic proteinase present in seeds cleaves Arabidopsis 2S albumin precursors in vitro. Journal of Biological Chemistry, 268: 20884-20891.
Da Silva, P., V. Eyraud, M. Carre-Pierrat, C. Sivignon, I. Rahioui, C. Royer and F. Gressent. 2012. High toxicity and specificity of the saponin 3-GlcA-28- AraRhaxyl-medicagenate, from Medicago truncatula seeds, for Sitophilus oryzae. BMC Chemical Biology, 12: 3.
Dalgalarrondo, M., J.-M. Robin and J.-L. Azanza. 1986. Subunit composition of the globulin fraction of rapeseed (Brassica napus L.). Plant Science, 43: 115-124.
Datta, A.B., G.L. Hura and C. Wolberger. 2009. The structure and conformation of Lys63-linked tetraubiquitin. Journal of Molecular Biology, 392: 1117-1124.
DeLano, W. 2002. The PyMOL Molecular Graphics System, Version 1.2 r3pre, Schrödinger, LLC. There is no corresponding record for this reference.
DeLisle, A.J. and M.L. Crouch. 1989. Seed storage protein transcription and mRNA levels in Brassica napus during development and in response to exogenous abscisic acid. Plant Physiology, 91: 617-623.
Page 114
Chapter 5 References
Delseny, M. and M. Raynal, 1999. Globulin storage proteins in crucifers and non-legume dicotyledonous families. Seed Protein, 427-451.
Depigny-This, D., M. Raynal, L. Aspart, M. Delseny and F. Grellet. 1992. The cruciferin gene family in radish. Plant Molecular Biology, 20: 467-479.
Dikic, I., S. Wakatsuki and K.J. Walters. 2009. Ubiquitin-binding domains—from structures to functions. Nature Reviews Molecular Cell Biology, 10: 659-671.
Downey, R.K., G. Robbelen and A. Ashri, 1989. Oil crops of the world: Their breeding and utilization McGraw-Hill.
Drewnowski, A. and C. Gomez-Carneros. 2000. Bitter taste, phytonutrients, and the consumer: a review. The American Journal of Clinical Nutrition, 72: 1424-1435.
Duong-Ly, K.C. and S.B. Gabelli. 2014. Chapter seven-salting out of proteins using ammonium sulfate precipitation. Methods In Enzymology, 541: 85-94.
Durner, J. and P. Böger. 1995. Ubiquitin in the prokaryote Anabaena variabilis. Journal of Biological Chemistry, 270: 3720-3725.
Castro, V.O. and L.P. Vernon. 1989. Hemolytic activity of thionin from Pyrularia pubera nuts and snake venom toxins of Naja naja species: Pyrularia thionin and snake venom cardiotoxin compete for the same membrane site. Toxicon, 27: 511-517.
Eddins, M.J., R. Varadan, D. Fushman, C.M. Pickart and C. Wolberger. 2007. Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH. Journal of Molecular Biology, 367: 204-211
Evans, P. 2006. Scaling and assessment of data quality. Acta Crystallographica Section D: Biological Crystallography, 62: 72-82.
Falk, A., J. Taipalensuu, B. Ek, M. Lenman and L. Rask. 1995. Characterization of rapeseed myrosinase-binding protein. Planta, 195: 387-395.
Fernie, A.R. and N. Schauer. 2009. Metabolomics-assisted breeding: a viable option for crop improvement? Trends in Genetics, 25: 39-48.
Page 115
Chapter 5 References
Flanders, A. and S. Abdulkarim. 1985. The composition of seed and seed oils of Taramira (Eruca sativa). Journal of the American Oil Chemists’ Society, 62: 1134-1135.
Florack, D. and W. Stiekema. 1994. Thionins: properties, possible biological roles and mechanisms of action. Plant Molecular Biology, 26: 25-37.
Franke, D. and D.I. Svergun. 2009. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. Journal of Applied Crystallography, 42: 342-346.
Fuentes, E., M. Alarcón, M. Fuentes, G. Carrasco and I. Palomo. 2014. A Novel Role of Eruca sativa Mill.(rocket) extract: Antiplatelet (NF-κB Inhibition) and antithrombotic activities. Nutrients, 6: 5839-5852.
Gardiner, J.B., M.J. Morra, C.V. Eberlein, P.D. Brown and V. Borek. 1999. Allelochemicals released in soil following incorporation of rapeseed (Brassica napus) green manures. Journal of Agricultural and Food Chemistry, 47: 3837- 3842.
Gehrig, P. and K. Biemann. 1995. Assignment of the disulfide bonds in napin, a seed storage protein from Brassica napus, using matrix-assisted laser desorption ionization mass spectrometry. Peptide Research, 9: 308-314.
Geng, F., S. Wenzel and W.P. Tansey. 2012. Ubiquitin and proteasomes in transcription. Annual Review of Biochemistry, 81: 177-201.
Ghafari, S., S. Esmaeili, F. Naghibi and M. Mosaddegh. 2013. Plants used to treat malaria like fever in Iranian traditional medicine. Intl J Trad Herb Med., 1: 168- 176.
Ghazanfar, S.A., 1994. Handbook of Arabian medicinal plants.CRC press.
Gillberg, L. and B. Törnell. 1976. Preparation of rapeseed protein. ln isolates. Dissolution and Precipitation Behavior of Rapeseed Proteins. Journal of Food Science, 41: 1063-1069.
Page 116
Chapter 5 References
Giudici, A.M., M.C. Regente, J. Villalaín, K. Pfüller, U. Pfüller and L. De La Canal. 2004. Mistletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi. Physiologia Plantarum, 121: 2-7.
Gressent, F., P. Da Silva, V. Eyraud, L. Karaki and C. Royer. 2011. Pea Albumin 1 subunit b (PA1b), a promising bioinsecticide of plant origin. Toxins, 3: 1502- 1517.
Gressent, F., I. Rahioui and Y. Rahbe. 2003. Characterization of a high‐affinity binding site for the pea albumin 1b entomotoxin in the weevil Sitophilus. European Journal of Biochemistry, 270: 2429-2435.
Guerena, M. 2006. Cole crops and other Brassicas: Organic production. A publication of ATTRA: 1-19.
Guex, N. and M.C. Peitsch. 1997. SWISS‐MODEL and the Swiss‐Pdb Viewer: an environment for comparative protein modeling. Electrophoresis, 18: 2714-2723.
Haglund, K. and I. Dikic. 2005. Ubiquitylation and cell signaling. The EMBO Journal, 24: 3353-3359.
Henderson, E. and R. Van En, 2007. Sharing the harvest: a citizen's guide to Community Supported Agriculture. Chelsea Green Publishing.
Herman, E.M. and B.A. Larkins. 1999. Protein storage bodies and vacuoles. The Plant Cell, 11: 601-613.
Hewick, R.M., M.W. Hunkapiller, L.E. Hood and W.J. Dreyer. 1981. A gas-liquid solid phase peptide and protein sequenator. Journal of Biological Chemistry, 256: 7990-7997.
Higdon, J.V., B. Delage, D.E. Williams and R.H. Dashwood. 2007. Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis. Pharmacological Research, 55: 224-236.
Higgins, T., P. Chandler, P. Randall, D. Spencer, L. Beach, R. Blagrove, A. Kortt and A. Inglis. 1986. Gene structure, protein structure, and regulation of the synthesis of a
Page 117
Chapter 5 References
sulfur-rich protein in pea seeds. Journal of Biological Chemistry, 261: 11124- 11130.
Hilker, M. and T. Meiners. 2002. Induction of plant responses to oviposition and feeding by herbivorous arthropods: a comparison. Entomologia Experimentalis et Applicata, 104: 181-192.
Hochstrasser, M. 2000. Evolution and function of ubiquitin-like protein-conjugation systems. Nature Cell Biology, 2: E153-E157.
Hokkanen, H.M. 1991. Trap cropping in pest management. Annual review of entomology, 36: 119-138.
Hou, A., K. Liu, N. Catawatcharakul, X. Tang, V. Nguyen, W.A. Keller, E.W. Tsang and Y. Cui. 2005. Two naturally occurring deletion mutants of 12S seed storage proteins in Arabidopsis thaliana. Planta, 222: 512-520.
Hu, M., P. Li, M. Li, W. Li, T. Yao, J.-W. Wu, W. Gu, R.E. Cohen and Y. Shi. 2002. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell, 111: 1041-1054.
Huang, A.H. 1992. Oil bodies and oleosins in seeds. Annual Review of Plant Biology, 43: 177-200.
Hughes, P., E. Dennis, M. Whitecross, D. Llewellyn and P. Gage. 2000. The cytotoxic plant protein, β-purothionin, forms ion channels in lipid membranes. Journal of Biological Chemistry, 275: 823-827.
Hura, G.L., H. Budworth, K.N. Dyer, R.P. Rambo, M. Hammel, C.T. McMurray and J.A. Tainer. 2013. Comprehensive macromolecular conformations mapped by quantitative SAXS analyses. Nature Methods, 10: 453-454.
Jacques, D.A., J.M. Guss and J. Trewhella. 2012. Reliable structural interpretation of small-angle scattering data from bio-molecules in solution-the importance of quality control and a standard reporting framework. BMC Structural Biology, 12: 9.
Page 118
Chapter 5 References
Jiang, L., T.E. Phillips, C.A. Hamm, Y.M. Drozdowicz, P.A. Rea, M. Maeshima, S.W. Rogers and J.C. Rogers. 2001. The protein storage vacuole. J Cell Biol., 155: 991- 1002.
Jin, L., A. Williamson, S. Banerjee, I. Philipp and M. Rape. 2008. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell, 133: 653-665.
Jin, R.-G., Y.-B. Liu, B. Tabashnik and D. Borthakur. 2000. Development of transgenic cabbage (Brassica oleracea var. capitata) for insect resistance by Agrobacterium tumefaciens-mediated transformation. In Vitro Cellular & Developmental Biology-Plant, 36: 231-237.
Johansson, S., J. Gullbo, P. Lindholm, B. Ek, E. Thunberg, G. Samuelsson, R. Larsson, L. Bohlin and P. Claeson. 2003. Small, novel proteins from the mistletoe Phoradendron tomentosum exhibit highly selective cytotoxicity to human breast cancer cells. Cellular and Molecular Life Sciences, 60: 165-175.
Johnson, K.A., E. Kim, M.M. Teeter, S.W. Suh and B. Stec. 2005. Crystal structure of α‐ hordothionin at 1.9 Å resolution. FEBS Letters, 579: 2301-2306.
Jouvensal, L., L. Quillien, E. Ferrasson, Y. Rahbé, J. Guéguen and F. Vovelle. 2003. PA1b, an Insecticidal Protein Extracted from Pea Seeds (Pisum sativum): 1H-2-D NMR Study and Molecular Modeling†. Biochemistry, 42: 11915-11923.
Jurado, S., S. Díaz‐Triviño, Z. Abraham, C. Manzano, C. Gutierrez and C.d. Pozo. 2008. SKP2A, an F‐box protein that regulates cell division, is degraded via the ubiquitin pathway. The Plant Journal, 53: 828-841.
Kabsch, W. 2010. Xds. Acta Crystallographica Section D: Biological Crystallography, 66: 125-132.
Kamadurai, H.B., J. Souphron, D.C. Scott, D.M. Duda, D.J. Miller, D. Stringer, R.C. Piper and B.A. Schulman. 2009. Insights into ubiquitin transfer cascades from a structure of a UbcH5B∼ ubiquitin-HECT NEDD4L complex. Molecular Cell, 36: 1095-1102.
Page 119
Chapter 5 References
Katavic, V., G.K. Agrawal, M. Hajduch, S.L. Harris and J.J. Thelen. 2006. Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars. Proteomics, 6: 4586-4598.
Khalequzzaman, M., M. Khatun and D. Talukdar. 1994. Growth of Tribolium confusum Duv. on wheat flour with various yeast levels. International Pest Control, 36: 128-130.
Khan, M., K. Javed and M. Hasnain Khan. 1991. Chemical constituents of the aerial parts of Sisymbrium irio. Journal of the Indian Chemical Society, 68.
Kim, S.-J. and G. Ishii. 2006. Glucosinolate profiles in the seeds, leaves and roots of rocket salad (Eruca sativa Mill.) and anti-oxidative activities of intact plant powder and purified 4-methoxyglucobrassicin. Soil Science and Plant Nutrition, 52: 394-400.
Kissen, R., J.T. Rossiter and A.M. Bones. 2009. The ‗mustard oil bomb‘: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system. Phytochemistry Reviews, 8: 69-86.
Komander, D., F. Reyes‐Turcu, J.D. Licchesi, P. Odenwaelder, K.D. Wilkinson and D. Barford. 2009. Molecular discrimination of structurally equivalent Lys 63‐linked and linear polyubiquitin chains. EMBO Reports, 10: 466-473.
Konarev, P.V., V.V. Volkov, A.V. Sokolova, M.H. Koch and D.I. Svergun. 2003. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. Journal of Applied Crystallography, 36: 1277-1282.
Krets, L., L. Domashenko, N. Negare and A. Railyan. 1987. Glycoside from Sisymbrium species. Ser. Biol. Khim. Nauk, 6: 25.
Krzyzaniak, A., T. Burova, T. Haertle and J. Barciszewski. 1998. The structure and properties of Napin‐seed storage protein from rape (Brassica napus L.). Food/Nahrung, 42: 201-204.
Page 120
Chapter 5 References
Kurosu, M., P. Narayanasamy, K. Biswas, R. Dhiman and D.C. Crick. 2007. Discovery of 1, 4-didydroxy-2-naphthoate prenyltransferase inhibitors: new drug leads for multidrug-resistant gram-positive pathogens. Journal of Medicinal Chemistry, 50: 3973-3975.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
Lange, O.F., N.-A. Lakomek, C. Farès, G.F. Schröder, K.F. Walter, S. Becker, J. Meiler, H. Grubmüller, C. Griesinger and B.L. De Groot. 2008. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science, 320: 1471-1475.
Larkin, R.P. and T.S. Griffin. 2007. Control of soilborne potato diseases using Brassica green manures. Crop Protection, 26: 1067-1077.
Lin, P. and T.B. Ng. 2008. A novel and exploitable antifungal peptide from kale (Brassica alboglabra) seeds. Peptides, 29: 1664-1671.
Liu, C.-W., C.-C. Lin, J.-C. Yiu, J.J. Chen and M.-J. Tseng. 2008. Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella. Theoretical and Applied Genetics, 117: 75-88.
Loeza-Ángeles, H., E. Sagrero-Cisneros, L. Lara-Zárate, E. Villagómez-Gómez, J.E. López-Meza and A. Ochoa-Zarzosa. 2008. Thionin Thi2. 1 from Arabidopsis thaliana expressed in endothelial cells shows antibacterial, antifungal and cytotoxic activity. Biotechnology Letters, 30: 1713.
Lönnerdal, B. and J.-C. Janson. 1972. Studies on Brassica seed proteins: I. The low molecular weight proteins in rapeseed. isolation and characterization. Biochimica et Biophysica Acta (BBA)-Protein Structure, 278: 175-183.
Love, C.G., A.J. Robinson, G.A. Lim, C.J. Hopkins, J. Batley, G. Barker, G.C. Spangenberg and D. Edwards. 2005. Brassica ASTRA: an integrated database for Brassica genomic research. Nucleic Acids Research, 33: D656-D659.
Page 121
Chapter 5 References
Malabat, C., H. Atterby, Q. Chaudhry, M. Renard and J. Guéguen, Year. Genetic variability of rapeseed protein composition. In, Vol. 1.
Manavalan, P. and W.C. Johnson. 1983. Sensitivity of circular dichroism to protein tertiary structure class.
Martínez-Sánchez, A., A. Marín, R. Llorach, F. Ferreres and M.I. Gil. 2006. Controlled atmosphere preserves quality and phytonutrients in wild rocket (Diplotaxis tenuifolia). Postharvest Biology and Technology, 40: 26-33.
Martin, M.M. and G.J. Wiederrecht. 1996. A filter-based, 96-well microtiter plate calcineurin phosphatase assay. Methods, 9: 155-159.
Matsumoto, M.L., K.E. Wickliffe, K.C. Dong, C. Yu, I. Bosanac, D. Bustos, L. Phu, D.S. Kirkpatrick, S.G. Hymowitz and M. Rape. 2010. K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody. Molecular cell, 39: 477-484.
Menegatti, E., S. Palmieri, P. Walde and P.L. Luisi. 1985. Isolation and characterization of a trypsin inhibitor from white mustard (Sinapis alba L.). Journal of Agricultural and Food Chemistry, 33: 784-789.
Menéndez-Arias, L., R.I. Monsalve, J. Gavilanes and R. Rodríguez. 1987. Molecular and spectroscopic characterisation of a low molecular weight seed storage protein from yellow mustard (Sinapis alba L.). International Journal of Biochemistry, 19: 899-907.
Menéndez‐Arias, L., I. Moneo, J. Domínguez and R. Rodríguez. 1988. Primary structure of the major allergen of yellow mustard (Sinapis alba L.) seed, Sin a I. European Journal of Biochemistry, 177: 159-166.
Monsalve, R.I. and R. Rodriguez. 1990. Purification and characterization of proteins from the 2S fraction from seeds of the Brassicaceae family. Journal of Experimental Botany, 41: 89-94.
Page 122
Chapter 5 References
Montesano, M., G. Brader and E.T. Palva. 2003. Pathogen derived elicitors: searching for receptors in plants. Molecular Plant Pathology, 4: 73-79.
Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65: 55-63.
Muench, S.P., S. Rawson, V. Eyraud, A.F. Delmas, P. Da Silva, C. Phillips, J. Trinick, M.A. Harrison, F. Gressent and M. Huss. 2014. PA1b inhibitor binding to subunits c and e of the vacuolar ATPase reveals its insecticidal mechanism. Journal of Biological Chemistry, 289: 16399-16408.
Mukhopadhyay, D. and H. Riezman. 2007. Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science, 315: 201-205.
Mulligan, E.A., N. Ferry, L. Jouanin, K.F. Walters, G.R. Port and A.M. Gatehouse. 2006. Comparing the impact of conventional pesticide and use of a transgenic pest‐ resistant crop on the beneficial carabid beetle Pterostichus melanarius. Pest Management Science, 62: 999-1012.
Muluye, A.B., E. Melese and G.M. Adinew. 2015. Antimalarial activity of 80% methanolic extract of Brassica nigra (L.) Koch.(Brassicaceae) seeds against Plasmodium berghei infection in mice. BMC Complementary and Alternative Medicine, 15: 367.
Müntz, K., 1998. Deposition of storage proteins. Plant Molecular Biology, 38: 77-99.
Murphy, D. 1990. Storage lipid bodies in plants and other organisms. Progress In Lipid Research, 29: 299-324.
Neumann, G.M., R. Condron and G.M. Polya. 1996a. Purification and sequencing of yellow mustard seed napin small and large chains that are phosphorylated by plant calcium-dependent protein kinase and are calmodulin antagonists. Plant Science, 119: 49-66.
Page 123
Chapter 5 References
Neumann, G.M., R. Condron, I. Thomas and G.M. Polya. 1996b. Purification and sequencing of multiple forms of Brassica napus seed napin small chains that are calmodulin antagonists and substrates for plant calcium-dependent protein kinase. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1295: 23-33.
Newkirk, R. 2009. Canola meal feed industry guide. Canola Council of Canada. 4th Edition, Winnipeg, Manitoba.
Nietzel, T., N.V. Dudkina, C. Haase, P. Denolf, D.A. Semchonok, E.J. Boekema, H.-P. Braun and S. Sunderhaus. 2013. The native structure and composition of the cruciferin complex in Brassica napus. Journal of Biological Chemistry, 288: 2238-2245.
Oard, S., M. Rush and J. Oard. 2004. Characterization of antimicrobial peptides against a US strain of the rice pathogen Rhizoctonia solani. Journal of Applied Microbiology, 97: 169-180.
Østergaard, J., P. Højrup and J. Knudsen. 1995. Amino acid sequences of three acyl- binding/lipid-transfer proteins from rape seedlings. Biochimica et Biophysica Acta (BBA)-Lipids and Lipid Metabolism, 1254: 169-179.
Otwinowski, Z. and W. Minor. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology, 276: 307-326.
Ozaki, Y., W. Keishiro, H. Matsubara, T. Nakanishi and H. Yoshizumi. 1980. Amino acid sequence of a purothionin homolog from barley flour. Journal of Biochemistry, 87: 549-555.
Pal, A., J.E. Debreczeni, M. Sevvana, T. Gruene, B. Kahle, A. Zeeck and G.M. Sheldrick. 2008. Structures of viscotoxins A1 and B2 from European mistletoe solved using native data alone. Acta Crystallographica Section D: Biological Crystallography, 64: 985-992.
Page 124
Chapter 5 References
Palomares, O., J. Cuesta-Herranz, A. Vereda, S. Sirvent, M. Villalba and R. Rodríguez. 2005. Isolation and identification of an 11S globulin as a new major allergen in mustard seeds. Annals of Allergy, Asthma & Immunology, 94: 586-592.
Patrick, M.H., 2007. Crystallography made crystal clear: A guide for users of macromolecular models. Wiley Online Library.
Pelegrini, P.B. and O.L. Franco. 2005. Plant γ-thionins: novel insights on the mechanism of action of a multi-functional class of defense proteins. The International Journal of Biochemistry & Cell biology, 37: 2239-2253.
Peng, J., D. Schwartz, J.E. Elias, C.C. Thoreen, D. Cheng, G. Marsischky, J. Roelofs, D. Finley and S.P. Gygi. 2003. A proteomics approach to understanding protein ubiquitination. Nature Biotechnology, 21: 921-926.
Perera, S.P., T.C. McIntosh and J.P. Wanasundara. 2016. Structural properties of cruciferin and napin of Brassica napus (Canola) Show Distinct Responses to Changes in pH and Temperature. Plants, 5: 36.
Petoukhov, M.V., D. Franke, A.V. Shkumatov, G. Tria, A.G. Kikhney, M. Gajda, C. Gorba, H.D. Mertens, P.V. Konarev and D.I. Svergun. 2012. New developments in the ATSAS program package for small-angle scattering data analysis. Journal of Applied Crystallography, 45: 342-350.
Pettersen, E.F., T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng and T.E. Ferrin. 2004. UCSF Chimera—a visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25: 1605-1612.
Pickart, C.M. 2001. Mechanisms underlying ubiquitination. Annual Review of Biochemistry, 70: 503-533.
Pickett, J., L. Wadhams and C. Woodcock. 1997. Developing sustainable pest control from chemical ecology. Agriculture, Ecosystems & Environment, 64: 149-156.
Page 125
Chapter 5 References
Potts, D.A., G.W. Rakow and D.R. Males. 1999. Canola-quality Brassica juncea, a new oilseed crop for the Canadian prairies. New Horizons for an old crop. Proc 10th Intl Rapeseed Congr, Canberra, Australia.
Powers, J.-P.S. and R.E. Hancock. 2003. The relationship between peptide structure and antibacterial activity. Peptides, 24: 1681-1691.
Praveen, K., P. Naga, K. Murali and M. Swarnalatha. 2013. Evaluation of antiepileptic activity of methanolic extract of Brassica nigra seeds in mice. IJPI., 3: 73-84.
Provencher, S.W. 1982. A constrained regularization method for inverting data represented by linear algebraic or integral equations. Computer Physics Communications, 27: 213-227.
Rahighi, S., F. Ikeda, M. Kawasaki, M. Akutsu, N. Suzuki, R. Kato, T. Kensche, T. Uejima, S. Bloor and D. Komander. 2009. Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation. Cell, 136: 1098-1109.
Rahioui, I., C. Laugier, S. Balmand, P. Da Silva, Y. Rahbe and F. Gressent. 2007. Toxicity, binding and internalization of the pea-A1b entomotoxin in Sf9 cells. Biochimie, 89: 1539-1543.
Rao, A.G. and M.N. Rao. 1979. Effect of sodium dodecyl sulphate on the 12S fraction of mustard proteins (B. juncea). Chemical Biology & Drug Design, 14: 300-306.
Rask, L., E. Andréasson, B. Ekbom, S. Eriksson, B. Pontoppidan and J. Meijer, 2000. Myrosinase: gene family evolution and herbivore defense in Brassicaceae Plant Molecular, 42: 93-113.
Reed, J. and V. Kinzel. 1984. Near-and far-ultraviolet circular dichroism of the catalytic subunit of adenosine cyclic 5'-monophosphate dependent protein kinase. Biochemistry, 23: 1357-1362.
Rehman, S., U.A. Ashfaq, S. Riaz, T. Javed and S. Riazuddin. 2011. Antiviral activity of Acacia nilotica against Hepatitis C Virus in liver infected cells. Virology Journal, 8: 220.
Page 126
Chapter 5 References
Riaño-Pachón, D.M., A. Nagel, J. Neigenfind, R. Wagner, R. Basekow, E. Weber, B. Mueller-Roeber, S. Diehl and B. Kersten. 2009. GabiPD: the GABI primary database—a plant integrative ‗omics‘ database. Nucleic Acids Research, 37: D954-D959.
Robin, J., V. Inquello, B. Mimouni and J. Azanza. 1991. Relationship between immunological properties and structural model of 11S rapeseed globulin. Phytochemistry, 30: 3511-3513.
Rödin, J. and L. Rask. 1990. The relationship between mature chains and their precursors of cruciferin, the 12S storage protein of Brassica napus. Plant Science, 70: 57-63.
Rödin, J., S. Sjödahl, L.-G. Josefsson and L. Rask. 1992. Characterization of a Brassica napus gene encoding a cruciferin subunit: estimation of sizes of cruciferin gene families. Plant Molecular Biology, 20: 559-563.
Romagnoli, S., F. Fogolari, M. Catalano, L. Zetta, G. Schaller, K. Urech, M. Giannattasio, L. Ragona and H. Molinari. 2003. NMR Solution Structure of Viscotoxin C1 from Viscum album Species Coloratum ohwi: Toward a Structure− Function Analysis of Viscotoxins. Biochemistry, 42: 12503-12510.
Sambrook, J., E.F. Fritsch and T. Maniatis, 1989. Molecular cloning: a laboratory manual Cold spring harbor laboratory press.
Schäfer, A., M. Kuhn and H. Schindelin. 2014. Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules. Acta Crystallographica Section D: Biological Crystallography, 70: 1311-1320.
Schmidt, I., D. Renard, D. Rondeau, P. Richomme, Y. Popineau and M.A.-V. Axelos. 2004. Detailed physicochemical characterization of the 2S storage protein from rape (Brassica napus L.). Journal of Agricultural and Food Chemistry, 52: 5995- 6001.
Schwenke, K., 1994. Rapeseed proteins New and developing sources of food proteins. Springer, pp. 281-306.
Page 127
Chapter 5 References
Schwenke, K., B. Raab, K.J. Linow, W. Pahtz and J. Uhlig. 1981. Isolation of the 12 S globulin from Rapeseed (Brassica napus L.) and characterization as a ―neutral‖ protein on seed proteins. Part 13. Molecular Nutrition & Food Research, 25: 271- 280.
Schwenke, K., B. Raab, P. Plietz and G. Damaschun. 1983. The structure of the 12S globulin from rapeseed (Brassica napus L.). Molecular Nutrition & Food Research, 27: 165-175.
Shafaghat, A. 2010. Phytochemical investigation of quranic fruits and plants. Journal of Medicinal Plants, 3: 61-66.
Shelton, A. and F. Badenes-Perez. 2006. Concepts and applications of trap cropping in pest management. Annu. Rev. Entomol., 51: 285-308.
Shelton, A., S. Hatch, J. Zhao, M. Chen, E. Earle and J. Cao. 2008. Suppression of diamondback moth using Bt-transgenic plants as a trap crop. Crop Protection, 27: 403-409.
Shewry, P.R., J.A. Napier and A.S. Tatham. 1995. Seed storage proteins: structures and biosynthesis. The Plant Cell, 7: 945.
Shih, S.C., K.E. Sloper‐Mould and L. Hicke. 2000. Monoubiquitin carries a novel internalization signal that is appended to activated receptors. The EMBO Journal, 19: 187-198.
Silverstein, K.A., M.A. Graham, T.D. Paape and K.A. VandenBosch. 2005. Genome organization of more than 300 defensin-like genes in Arabidopsis. Plant Physiology, 138: 600-610.
Simoes, M., R.N. Bennett and E.A. Rosa. 2009. Understanding antimicrobial activities of phytochemicals against multidrug resistant bacteria and biofilms. Natural Product Reports, 26: 746-757.
Sims, R.P. 1971. Edible protein products from Cruciferae seed meals. Journal of the American Oil Chemists' Society, 48: 733-736.
Page 128
Chapter 5 References
Sjödahl, S., J. Rödin and L. Rask. 1991. Characterization of the 12S globulin complex of Brassica napus. European Journal of Biochemistry, 196: 617-621.
Sloper-Mould, K.E., J.C. Jemc, C.M. Pickart and L. Hicke. 2001. Distinct functional surface regions on ubiquitin. Journal of Biological Chemistry, 276: 30483-30489.
Smith, B.J., J.A. Kirkegaard and G.N. Howe. 2004. Impacts of Brassica break-crops on soil biology and yield of following wheat crops. Crop and Pasture Science, 55: 1- 11.
Snapp, S., K. Date, K. Cichy and K. O‘Neil. 2006. Mustards—A Brassica cover crop for Michigan. Extension Bulletin E-2956. Michigan State Univ.
Snowdon, R. and W. Friedt. 2004. Molecular markers in Brassica oilseed breeding: current status and future possibilities. Plant Breeding, 123: 1-8.
Snowdon, R., W. Lühs and W. Friedt, 2007. Oilseed rape Oilseeds. Springer, pp. 55-114.
Soulier, J. 1994. Oil content of some Sismbrium plants. Rev. Fr. Crops. gras, 41: 73.
Stec, B., O. Markman, U. Rao, G. Heffron, S. Henderson, L. Vernon, V. Brumfeld and M. Teeter. 2004. Proposal for molecular mechanism of thionins deduced from physico‐chemical studies of plant toxins. Chemical Biology & Drug Design, 64: 210-224.
Stein, A.C., M. Sortino, C. Avancini, S. Zacchino and G. von Poser. 2005. Ethnoveterinary medicine in the search for antimicrobial agents: Antifungal activity of some species of Pterocaulon (Asteraceae). Journal of Ethnopharmacology, 99: 211-214.
Strobl, G.R., 1997. The physics of polymers: concepts for understanding their structures and behavior Springer Verlag.
Svergun, D., C. Barberato and M.H. Koch. 1995. CRYSOL–a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. Journal of Applied Crystallography, 28: 768-773.
Page 129
Chapter 5 References
Svergun, D.I., M.H. Koch, P.A. Timmins and R.P. May, 2013. Small angle X-ray and neutron scattering from solutions of biological macromolecules Oxford University Press.
Tandang-Silvas, M.R.G., T. Fukuda, C. Fukuda, K. Prak, C. Cabanos, A. Kimura, T. Itoh, B. Mikami, S. Utsumi and N. Maruyama. 2010. Conservation and divergence on plant seed 11S globulins based on crystal structures. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1804: 1432-1442.
Teeter, M.M., X.Q. Ma, U. Rao and M. Whitlow. 1990. Crystal structure of a protein‐ toxin α1‐purothionin at 2.5 Å and a comparison with predicted models. Proteins: Structure, Function, and Bioinformatics, 8: 118-132.
Tenno, T., K. Fujiwara, H. Tochio, K. Iwai, E.H. Morita, H. Hayashi, S. Murata, H. Hiroaki, M. Sato and K. Tanaka. 2004. Structural basis for distinct roles of Lys63‐ and Lys48‐linked polyubiquitin chains. Genes to Cells, 9: 865-875.
Terras, F.R., S. Torrekens, F. Van Leuven, R.W. Osborn, J. Vanderleyden, B.P. Cammue and W.F. Broekaert. 1993. A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species. FEBS Letters, 316: 233-240.
Thomma, B.P., B.P. Cammue and K. Thevissen. 2003. Mode of action of plant defensins suggests therapeutic potential. Current Drug Targets-Infectious Disorders, 3: 1-8.
Thompson, J.D., D.G. Higgins and T.J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22: 4673-4680.
Thrower, J.S., L. Hoffman, M. Rechsteiner and C.M. Pickart. 2000. Recognition of the polyubiquitin proteolytic signal. The EMBO Journal, 19: 94-102.
Tomar, R.S. and V. Shrivastava. 2014. Efficacy evaluation of ethanolic extract of Brassica nigra as potential antimicrobial agent against selected microorganisms. IJPHC., 3: 117-123.
Page 130
Chapter 5 References
Towbin, H., T. Staehelin and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, 76: 4350-4354.
Traka, M.H. and R.F. Mithen. 2011. Plant science and human nutrition: challenges in assessing health-promoting properties of phytochemicals. The Plant Cell, 23: 2483-2497.
Varadan, R., M. Assfalg, A. Haririnia, S. Raasi, C. Pickart and D. Fushman. 2004. Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling. Journal of Biological Chemistry, 279: 7055-7063.
Varadan, R., O. Walker, C. Pickart and D. Fushman. 2002. Structural properties of polyubiquitin chains in solution. Journal of Molecular Biology, 324: 637-647.
Venkatesh, A. and A.A. Rao. 1988. Isolation and characterization of low molecular weight protein from mustard (Brassica juncea). Journal of Agricultural and Food Chemistry, 36: 1150-1155.
Verhoeven, D.T., R.A. Goldbohm, G. van Poppel, H. Verhagen and P.A. van den Brandt. 1996. Epidemiological studies on brassica vegetables and cancer risk. Cancer Epidemiology and Prevention Biomarkers, 5: 733-748.
Vijay-Kumar, S., C.E. Bugg and W.J. Cook. 1987. Structure of ubiquitin refined at 1.8 Åresolution. Journal of Molecular Biology, 194: 531-544.
Virdee, S., Y. Ye, D.P. Nguyen, D. Komander and J.W. Chin. 2010. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nature Chemical Biology, 6: 750-757.
Vohora, S., S. Naqvi and I. Kumar. 1980. Antipyretic, analgesic and antimicrobial studies on Sisymbrium irio. Planta Medica, 38: 255-259.
Page 131
Chapter 5 References
Wan, L., A.R. Ross, J. Yang, D.D. Hegedus and A.R. Kermode. 2007. Phosphorylation of the 12S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds. Biochemical Journal, 404: 247-256.
Wanasundara, J.P. 2011. Proteins of Brassicaceae oilseeds and their potential as a plant protein source. Critical Reviews in Food Science and Nutrition, 51: 635-677.
Wang, J., Z. Chen, J. Du, Y. Sun and A. Liang. 2005. Novel insect resistance in Brassica napus developed by transformation of chitinase and scorpion toxin genes. Plant Cell Reports, 24: 549-555.
Weeks, S.D., K.C. Grasty, L. Hernandez‐Cuebas and P.J. Loll. 2009. Crystal structures of Lys‐63‐linked tri‐and di‐ubiquitin reveal a highly extended chain architecture. Proteins: Structure, Function, and Bioinformatics, 77: 753-759.
Wu, J. and A. Muir. 2008. Comparative structural, emulsifying, and biological properties of 2 major canola proteins, cruciferin and napin. Journal of Food Science, 73: C210-C216.
Yang, J.T., C.-S.C. Wu and H.M. Martinez. 1986. Calculation of protein conformation from circular dichroism. Methods in Enzymology, 130: 208-269.
Yehuda, H., Y. Soroka, M. Zlotkin-Frušić, A. Gilhar, Y. Milner and S. Tamir. 2012. Isothiocyanates inhibit psoriasis-related proinflammatory factors in human skin. Inflammation Research, 61: 735-742.
Page 132
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