Molecular Studies on Plant Based Cyclotides for Protein- Protein Interaction By Zahid Mushtaq M.Phil. (UAF) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2016 DECLARATION I hereby declare that the contents of the thesis, “Molecular studies on plant based cyclotides for protein-protein interaction” are product of my own research and no part has been copied from any published source (except the references, standard mathematical or genetic models/equations/formulae/protocols etc). I further declare that this work has not been submitted for award of any diploma/degree. The University may take action if the information provided is found inaccurate at any stage. Zahid Mushtaq 2005-ag-238 The Controller of Examinations, University of Agriculture, Faisalabad. “We the Supervisory Committee, certify that the contents and form of thesis submitted by Zahid Mushtaq, Regd. No. 2005-ag-238, have been found satisfactory and recommend that it be processed for evaluation, by the External Examiner (s) for the award of degree”. SUPERVISORY COMMITTEE 1. CHAIRMAN (Prof. Amer Jamil) 2. MEMBER (Prof. Tahira Iqbal ) 3. MEMBER (Dr. Nisar Ahmed) ACKNOWLEDGMENT Words are bound and knowledge is limited to praise Allah, the omnipotent, the beneficent and merciful. Peace and blessings be upon Holy Prophet Muhammad (SAW), the everlasting source of guidance and knowledge for humanity. With genuine humanity, I acknowledge your aid, God. Please bless this work with your acceptance. I have a pleasure to ensure my sincere gratitude and deepest thanks to Prof. Amer Jamil, whose stimulating supervision, guidance and support made this work possible. I heartily thank him very much for his valuable help and for his kindness. I express my gratitude to Dr. Julio A Camarero, who guided me to the fascinating world of cyclotides during my research work in University of Southern California, Los Angeles, CA, USA. His support, encouragement, inspiring attitude and enthusiasm were impressive during IRSIP, HEC scholarship. The support, help and company of the whole group are appreciated, especially Dr. Jagadish Khrishnappa and Dr. Teshome for their help in research work. I wish to thank Prof. Tahira Iqbal, Department of Biochemistry and Dr. Nisar Ahmad, Centre of Agricultural Biochemistry and Biotechnology (CABB) for their guidance, encouragement and help throughout my PhD studies. I am also thankful to my genius friends and fellows Ghulam Mustafa, Muhammad Naeem, Dr. Kashif Jilani and Dr. Muhammad Shahid for their love providing amenities and friendship. Many thanks are due to my dearest family for giving me so much joy and happiness outside lab. Special thanks to my brothers M. Awais, Shahid Mushtaq and all my beloved near ones for their prayers, guidance and best wishes at each and every fraction of my life. I owe immense feelings of love and thanks for my affectionate mother and father, as their prayers are always behind my each success and my loving sisters, my fiancy, in laws and bhabhies for their continuous encouragement, untiring efforts and their patience while I did this work. Zahid Mushtaq TABLE OF CONTENTS Sr. No. TITLE Page No. Chapter 1 Introduction 1-9 1.1. Bioactive peptides and proteins 1 1.2. Cyclic peptides and cyclotide 2 1.3. Sources and distribution of bioactive cyclotides 3 1.4. Structural features of Cyclotide 3 1.5. Genetics of cyclotides 4 1.6. Candidates of enzymatic synthesis of cyclotides in plants 5 1.7. Production of recombinant Cyclotide 6 1.8. Labeling cyclotide using unnatural amino acid Uaa 7 1.9. Research objectives 8 Chapter 2 Review of literature 10-51 2.1. History of peptide 10 2.2. Cyclic peptides as stable bioactive compounds 10 2.3. Types of Cyclic peptides/proteins 12 2.4. Sources of cyclic peptides 14 2.4.1. Terrestrial plants 15 2.4.2. Marine plant 16 2.5. Microbial cyclic antimicrobial peptides 16 2.6. Food derived peptides 17 2.6.1 Other examples and functions of cyclic peptides 18 2.7. Cyclotides 21 2.7.1. Discovering Cyclotides 21 2.7.2. Structure of cyclotides 23 2.7.3. The Möbius, bracelet and trypsin inhibitor cyclotides 25 2.7.4. Cyclotide isolations 26 2.7.5. Cyclotides bioactive action and Mechanisms 31 A bioinformatics based approach for Cyclotides variants 2.8. 35 and features 2.8.1. CyBase 36 2.9. Candidate Genes invovlved in cyclotide synthesis 37 2.9.1. Asparginyl Endopeptidases (AEP) 37 2.9.2. Protein Disulphide Isomerases (PDI) 38 2.10. Production strategies of Cyclotide 40 2.10.1. Cyclotide expression in yeast cells 41 Engineered scaffold of cyclotides with grafted epitopes for 2.10.2. 42 enhanced bioactive role 2.11. Labeling of Cyclotide 43 Incorporation of non-natural amino acids by orthogonal t-RNA 2.11.1. 43 in peptides 2.11.2. Click chemistry for proteins labeling 45 2.11.3. Labeling Cyclotides 47 i 2.12. The Future of peptide-based drugs 50 Chapter 3 Materials and methods 52-78 SECTION-I 52 3.1. Screening of plant extracts and cyclotide for bioactive potential 3.1.1. Selection of plant material 52 3.1.2. Preparation of plants extracts 53 3.1.3. Proteinase K treatment 53 3.1.4. Protein estimations 53 3.2. Antioxidant studies 53 3.2.1. Total Phenolic contents (TPC) 54 3.2.2. Total Flavonoid contents (TFC) 54 3.2.3 Reducing power assay 55 3.2.4. DPPH scavenging assay 55 3.3. Determination of DNA damaging protection activity 56 Evaluation of antimicrobial activity crude extract and its 3.4. 57 polar fractions 3.5. Formation and hydrolysis of biofilm potential 58 Cytotoxicity of plant extracts/MCOTI-I by Hemolytic 3.6. 59 Activity Evaluation of thrombolytic activity of protein/peptide 3.7. 60 extract 3.8. Ames test or mutagenicity test 60 SECTION-II 3.9. 63 Cyclotide Genes isolation studies 3.9.1. DNA isolation 63 3.9.2. PCR amplification 63 3.9.3. PCR product extraction and purification 65 3.9.4. Plasmid DNA isolations 65 3.9.5. Sequencing and bioinformatics analysis of the sequences 65 SECTION-III (completed in USC, Los Angeles, USA) 3.10. 66 Fluorescent labeling of cyclotide MCOTI-I proteins for studying protein-protein interactions 3.10.1. Instrumentation and materials used 66 3.11. Synthesis of DBCO-AMCA 67 3.12. Cloning of E. coli expression plasmids 67 3.13. Bacterial expression and purification 69 3.14. Expression and purification of intein precursor 1b 70 ii Invitro labeling of Texas red Succinamyl ester with Lys- 3.15. 72 MCOTI-I 3.16. In vitro labeling of MCoTI-AziF with DBCO-TxRD 73 3.17. Expression of trypsin-S195A-EGFP 73 Measurement of affinity constant between trypsin and 3.18. 74 TxRD-labeled MCoTI-AziF Western blot based optimization induction conditions of 3.19. 74 inteins expression for MCOTI-I production 3.20. Expression of cyclotide MCOTI-I in yeast cells (Published). 76 Results and discussions Section –I Chapter 4 79-174 Bioactive potential of indigenous cyclotide bearing plants along with MCOTI-I. Extraction of plant extracts with proteins and other 4.1. 80 photochemical using two different buffer systems. 4.1.1 Protein estimation by Bradford method 82 4.2. In vitro antioxidant potential of selected medicinal plants 83 4.2.1. Total phenolic contents (TPC) 83 4.2.2. Total flavonoids contents (TFC) 85 4.2.3. Reducing power assay 86 4.2.4. DPPH radical scavenging assay 88 4.2.5. DNA damage protection assay 90 4.3. Antimicrobial activity of plant extract 93 4.4. Biofilm formation inhibition/hydrolysis 98 4.5. In-vitro cytotoxicity assay 100 4.6. Mutagenicity assay by Ames test 102 4.8. Thrombolytic assay 105 Section –II 108 4.9. Isolation of Cyclotide genes from selected local plants Section – III (completed in USC, Los Angeles, USA) 4. 10. 117 Labeling of MCOTI-I cyclotide with florescent dye Texas Red for studying protein-protein interaction studies HPLC analysis of Cu++ free click chemistry Reaction of Texas 4.10.1. 120 red and DBCO amine and Texas Red DBCO amine iii 4.11. Cloning and expression of wild type MCOTI-I 123 Labeling of MCOTI-I-AziF with Texas Red for protein- 4.12. 132 protein interaction studies Labeling of wild type (WT) MCOTI-I-Lys-NH with Texas 4.13. 2 138 Red succinimyl ester Preparation of trypsin-sepharose beads for capturing MCOTI-I 4.13.1. 141 or MCOTI-I-AziF Optimization and HPLC Monitoring of Cu ++ free click reaction 4.14.1 148 in buffered Guandium-HCl Mass spectrophotometric analysis of each reactant and product 4.14.2. of azido and alkyne reaction for optimization of buffered 151 GdmHCl in future labeling MCOTI-I-AziF Expression and quantification of MCOTI-I-AziF at large scale 4.14.3. 153 for labeling reaction after optimization steps 4.14.4. Labeling reaction of MCOTI-I-AziF : Texas Red-DBCO 159 HPLC Purification and M/S confirmation of labelled peptide 4.14.5. 161 MCOTI-I-Azide-TxRD 4.14.6. Protein-protein interaction studies based on FRET analysis. 162 Expression optimization of Wild Type MCOTI-I 4.15. 168 production (Wester blot analysis) Expression of Cyclotide MCOTI-I in yeast cells (Published, 4.16. 169 Jagadish et al., 2015) Summary 175-177 Literature cited 178-200 Different antioxidant Activities of leaf and seed extracts of Appendix-I selected medicinal plants prepared in PBS and protein 201-202 extraction buffer with and without proteinase K treatment. Antibacterial and biofilm formation inhibition activity of different plants extracts prepared in Protein extraction buffer PEB (with and without pretreatment of Proteinase K enzyme), Appendix- Phosphate buffer saline PBS and purified