DESIGN, STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF HUMAN BETA DEFENSIN ANALOGS BALAKRISHNA CHANDRABABU KARTHIK (M. Sc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE DEPARTMENT OF CHEMISTRY, NATIONAL UNIVERSITY OF SINGAPORE 2009 This dissertation is dedicated to my beloved family (my Dad, my Mom, and my brothers Mr. Kumaran and Mr. Sakthivel) for their love and never-ending support i ACKNOWLEDGEMENTS I am very much grateful to National University of Singapore for providing me the environment, facilities and full support to carry out my graduate study. I feel very proud to address myself as a student of A/P Dr. Yang Daiwen; I have never seen such a humble, polite and gentle person with stunning intelligence. I would express my greatest gratitude to him for his kind guidance and directions throughout my research. His courteous nature made my research career more peaceful and fruitful. Further I thank A/P Dr. Henry Mok for all his valuable suggestions during our group meetings, his guidance and comments helped me a lot. I am grateful to Dr. Thorsten and Prof. Ding for examining me with their supportive suggestions during my PhD qualifying examination. It would be my pleasure to thank Prof. Ho Bow for his kind project collaboration and advices that helped us to publish our work. I also thank Mr. Ng Han Chong, the technical staff in Prof Ho’s lab, for his assistance in my bio-assay experiments. I highly appreciate A/P Dr. Swami and Prof. Kini for providing me several career advices throughout my course. I express my appreciation for the sincere help provided to me by Drs. Vivek, Sivaraman, Fan, Dip and Anirban. I thank the members of Lab1 (Drs. Siew Leong, Yvonne, Chiradip and Janarthanan) and my lab members (Zhang Jingfeng, Xu YingXi, Jia Jinghui, Yong Yeeheng, Lim Jack Wee, Zhi Lin, Long Dong, Huang Weidong, Zheng Yu, Zhou Zhimming, Xiaogang, Meng Dan, Yang Shuai, Wei Dahai, Iman Fahim Hameed, Wang Shujing, Liang Chen and Dai Xuhui) for all there help and support. Especially I thank deeply Madam Jingfeng who initiated my research in this lab by teaching many experimental tactics and treating me like her own younger brother. ii I thank Dr. Song for allowing me use his lab’s HPLC machine and my special thanks also goes to Dr. Liu Jingxian for his kind help in many of my experiments. People like Dr. Robin and Dr. Dilip G. R (Prof. Kini’s lab) and Guo Lin (Dr. Thorsten’s lab) also helped me in several ways for my progress and I highly value their help. I take this opportunity to thank my dear cousin brother and my local guardian Mr. K. Karthikeyan and with his wife Mrs. Sureka Karthikeyan for their moral support, advices and suggestions throughout my research career. I appreciate my friends and roommates; Venkatesh, Jayaprakash, Thangavelu, Thirumal, Balaji, Jothibasu and Manjeet who really shared my happiness and sorrow throughout my stay in Singapore. I declare my special thanks to all my friends (Tan, Sang, Rishi, Yee Heng, Jack, Wanlong, Shaveta, Toan, Shenbaga Moorthy and all others) in the Structural Biology Corridor whose presence made my work environment more comfortable. I appreciate K. R. Santosh Kumar and Vadivukarasi Raju for providing me a pleasing company during my first year of stay in Singapore which made my life more cheerful during that time. Last but not least, I can never use mere words to appreciate all my family members because without their love and incredible support I could not have accomplished anything in my life. iii TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iv SUMMARY ix LIST OF TABLES xii LIST OF FIGURES xiii LIST OF ABBREVIATIONS xviii CHAPTER INTRODUCTION 1.1 Antimicrobial peptides (AMPs) 1.1.1 Discovery and the timeline 1.1.2 Classifications of AMPs 1.1.2.1 Anionic peptides 1.1.2.2 Small cationic peptides 1.1.2.3 Cationic peptides rich in some specific amino acid groups 1.1.2.4 Disulfide bonded anionic and cationic peptides 1.1.2.5 Fragments of larger proteins 1.1.3 The family of defensins 1.1.3.1 Expression of defensins 11 1.1.3.2 Expression of Human Beta Defensins 11 1.1.4 Human beta defensin-3 12 1.2 Biological properties of defensins 15 iv 1.2.1 Antimicrobial activity 15 1.2.2 Chemotactic activity 18 1.3 The unresolved problems of HBD-3 18 1.4 Rationale behind the design of defensin mutations 19 1.5 General Methods for Characterizing Antimicrobial Peptides 21 1.5.1 Bio-assay techniques 21 1.5.1.1 Micro-dilution assay 22 1.5.1.2 Radial diffusion assay 22 1.5.2 Biophysical techniques 23 1.5.2.1 CD spectroscopy- Secondary and tertiary structural studies 23 1.5.2.2 Fluorescence spectroscopy- study of tertiary structural changes 24 1.5.2.3 Isothermal Titration Calorimetric binding studies 26 1.5.2.4 NMR techniques- For the determination of solution structures and dynamics 27 1.5.2.4.1 NMR phenomenon 28 1.5.2.4.2 Basic NMR parameters 28 1.5.2.4.3 Chemical shift 28 1.5.2.4.4 Scalar or J coupling 29 1.5.2.4.5 Relaxation 29 1.5.2.4.6 Nuclear Overhauser Effect (NOE) 30 1.5.2.4.7 The advantages and limitations of solution NMR for structural studies 30 1.5.2.4.8 General strategy of NMR structure determination 31 1.5.2.4.9 Sample preparation 31 v 1.5.2.4.10 Acquisition of NMR spectra 32 1.5.2.4.11 Resonance assignments 32 1.5.2.4.12 Restraint collection 33 1.5.2.4.13 Structure calculation and refinement 33 1.6 Our investigations to address unresolved problems with HBD-3 CHAPTER 34 MATERIALS AND METHODS 2.1 Design and molecular cloning 38 2.1.1 Protocol followed for the molecular cloning 2.2 Expression and purification of Def-A and rHBD-3 in LB and M9 media 39 44 2.2.1 Media (LB and M9) used for the expressions 44 2.2.2 Expression of peptides in M9 media 44 2.2.3 Purification of peptides 44 2.3 Tris-Tricine Protein/Peptide Separation Gels 45 2.4 Bactericidal assays 46 2.4.1 Hancock’s colony count assay 46 2.4.2 Radial diffusion assay 47 2.5 Interaction of Def-A with lipid vesicles, micelles and helix inducing solvents 48 2.5.1 Preparation of large unilamellar vesicles 48 2.5.2 CD spectroscopy: Strategy and acquisition 49 2.5.3 Isothermal Titration Calorimetry 49 2.5.4 Fluorescence emission spectroscopy 50 2.5.5 NMR spectroscopy: Data acquisition, processing, assignment and Structure determination 50 vi 2.5.6 Relaxation Study 52 2.5.7 Structure Calculation 52 2.5.8 Relaxation Data Analysis 53 2.5.9 Docking of Def-A with LPS 54 CHAPTER STRUCTURE AND DYNAMICS OF DEF-A BOUND TO SDS AND ITS BACTERICIDAL ACTIVITY 3.1 Design, expression and purification of Def-A and rHBD-3 56 3.1.1 Rationale behind the design of mutant Def-A 56 3.1.2 Expression and purification of Def-A and r-HBD-3 57 3.2 Activity against bacterial strains 60 3.3 Conformational changes upon binding to model membranes. 65 3.4 Isothermal Titration Calorimetry shows the binding of Def-A with POPG vesicles 68 3.5 NMR Studies of Def-A in SDS Bound State. 70 3.6 Relaxation parameters of Def-A. 79 CHAPTER LIPID SPECIFICITIES OF ‘DEF-A’ AND THE INTERACTION OF DEF-A WITH LPS 4.1 Lippopolysaccharide (LPS) Molecules of Gram Negative Bacteria 83 4.2. The toxic nature of LPS; the need for their sequestration 86 4.3. HBD-3 interacts specifically to LPS molecules 87 4.4 Isothermal Titration Calorimetric studies for binding of Def-A with LPS 88 4.5 Tr-NOE derived solution structure of LPS bound Def-A 90 4.6 Proposed model of Def-A/LPS complex 94 vii 4.7 Detergents and lipids used for the study 96 4.7.1 Comparison between SDS micelle and POPG vesicles 96 4.7.2 Preferential binding of Def-A towards lipids 101 CHAPTER CONCLUSION AND FUTURE WORK REFERENCES 105 108 viii SUMMARY Defensins comprises a large family of antimicrobial peptides and is classified in to three different sub-families (α, b and q defensins). Most of them are cationic, small, amphiphilic with fewer than 100 amino acids showing activity against vast number of deadly microorganisms like bacteria, fungi and enveloped viruses. Defensins are seen in all humans, animals, plants and insects. These peptides are known to display both cationic and hydrophobic surfaces on their structures which are considered to be the prerequisite for their ability to disrupt bacterial membranes. Among all the human defensins, Human Beta Defensin - (HBD-3) is known to exhibit many interesting behaviors including its unusually high positive charge (+11), broad spectrum of activity with comparatively low salt sensitivity etc. HBD-3 is potent against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecium, Streptococcus pneumoniae, Staphylococcus carnosus, and many others. It also has low lytic activity on the human erythrocytes and shows no cytotoxic effect against human cells. The enormous number of investigations regarding the activity and selectivity of HBD-3 shows that the membrane induced helix in HBD-3 might be important for its selection of bacterial membranes. In addition the correlation of activities with the structural changes of HBD-3 demonstrates the importance of the overall positive charge distribution and the hydrophobicity of HBD-3 on its activity and cytotoxicity. There are many findings on various aspects of HBD-3 but still only little is known about the influence of the structural properties (most importantly the S-S linkages) of the b-defensin on its direct interactions with eukaryotic membranes. The NMR ix Even though we have analyzed the Def-A behavior by considering various aspects, we lack experimental information regarding how the structurally folded wild type HBD-3 interacts in similar conditions. In fact, we could not study the direct properties of wild type HBD-3 because we faced several problems in expressing the wild type HBD-3 in its correctly folded form or oxidize the rHBD-3. Hence the future of this research should be focused on experimenting the native peptide’s properties to find out the direct structurefunction evidence for the activity mechanism of HBD-3. Designing several other analogues with mutations at positions other than the cysteine residues can be considered to localize the direct interaction or importance of any specific region in their activities. Though model membranes are considered to mimic the real membranes, they are mere speculations. Real biological membranes are very complicate in their composition when compared to any membrane mimicking medium. As they are comparable in charge and hydrophobicity, the study of interaction of antimicrobial peptide with model membranes is very common in terms of their efficiency. But in order to determine the behavior of peptides or proteins when they are inside a cell, the In-cell NMR techniques (Serber et al., 2001) are currently used and have gained much interest among the researchers. 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(HBD-3) and q (RTD-1) defensin family showing their sequences and specific pattern of disulfide connectivities 8 Ribbon diagrams of Selected Defensins A comparison of structures of different types of defensins (A) Human Neutrophil Peptide 3 (Dimer), (B) Insect defensin A, (C) Human Beta Defensin- 3 and (D) Rhesus Theta defensin (Ganz et al., 2003) 10 Primary sequence of HBD-3 showing the helix and beta. .. phagocytin and CAP molecules of rabbit and human were reported in 1985 (Selsted et al., 1985a and 1985b) and then recognized as natural peptide antibiotics and renamed as defensins (Ganz et al., 1985) The expression level and the identity of different classes of defensins differ among each species Until now both alpha and beta classes have been found in humans and rodents whereas in rabbits and guinea... -lactalbumin, human haemoglobin, lysozyme and ovalbumin Lactoferricin from lactoferrin 1.1.3 The family of defensins Defensins are wide spread and involved in the defense mechanisms of many organisms (hence the name defensins), and form the major class of AMPs next to the cathelicidins Cathelicidins differ from defensins in their structure and evolution (Zenetti et al., 1995; Lehrer et al., 2002) Defensins... rabbits and guinea pigs, only alpha defensins have been reported to be present in their leukocytes (Ganz et al., 1994) In cattle, sheep and pigs, only beta defensins have been reported so far (Huttner et al., 1999) The expression of q defensins has been shown in neutrophils and monocytes of only the rhesus monkeys 1.1.3.2 Expression of Human Beta Defensins The human beta defensins (HBDs) are either constitutively... profiles and ESI-MS determination (A) and (B) Shows the elution profiles of Def-A and rHBD-3 when loaded into a Waters RP-C18 analytical column and eluted using acetonitrile gradient Arrow heads indicate the peaks of Def-A and rHBD-3 (C) and (D) shows the ESI-ms analysis of unlabeled Def-A and rHBD-3 respectively with a series of multiply charged ions corresponding to the homogeneous peptides (E) and. .. A, (C) Human Beta Defensin- 3 and (D) Rhesus Theta defensin (Ganz et al., 2003) 10 1.1.3.1 Expression of defensins As stated before, defensins are widely expressed in almost all mammals, birds, invertebrates and plants Some recent reports show the presence of defensin even in the ebony-cup fungi (Schnorr et al., 2003) In mammals, the alpha defensins are generally expressed in the neutrophils and paneth... models and experimental proofs to demonstrate the possibility of dimer or oligomer formation (Pardi et al., 1992; Wimley et al., 1994) for some of the peptides but their significance in antimicrobial activity remain to be determined 9 Figure 1.3: Ribbon diagrams of Selected Defensins A comparison of structures of different types of defensins (A) Human Neutrophil Peptide 3 (Dimer), (B) Insect defensin. .. bonds The cationicity of defensins vary widely from +2 to +11 but they are generally known to have a balanced distribution of hydrophobic and charged amino acids Figure 1.2: Sequences and disulfide connectivites of defensins Representative peptides from a (HNP-1), b (HBD-3) and q (RTD-1) defensin family showing their sequences and specific pattern of disulfide connectivities 8 Despite of having low sequence... is due to existence of the amphiphillic structure and determined by the extent of distribution of cationic and hydrophobic regions on the peptide surface (Kluver et al., 2005) Even though little is known about the influence of the structural properties of the b -defensin on its interaction with eukaryotic membranes (Schneider et al., 2005), the presence and positioning of S-S bonds and N-terminal sequence... Despite of having low sequence homology the tertiary structures of a and b defensins are very similar, both having three stranded antiparallel b-sheets with the distinctive defensin fold but a short N-terminal alpha helix is seen only in human beta defensins The q defensins are dissimilar to a and b by having a circular backbone consisting of 18 amino acids (Figure 1.2) Even though the disulfide connectivities . diagrams of Selected Defensins. A comparison of structures of different types of defensins (A) Human Neutrophil Peptide 3 (Dimer), (B) Insect defensin A, (C) Human Beta Defensin- 3 and (D) Rhesus. DESIGN, STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF HUMAN BETA DEFENSIN ANALOGS BALAKRISHNA CHANDRABABU KARTHIK (M. Sc) . bonded anionic and cationic peptides 6 1.1.2.5 Fragments of larger proteins 7 1.1.3 The family of defensins 7 1.1.3.1 Expression of defensins 11 1.1.3.2 Expression of Human Beta Defensins