Design and characterization of functional novel oligopeptides

DESIGN AND CHARACTERIZATION OF FUNCTIONAL NOVEL OLIGOPEPTIDES ONG BOON TEE (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOE THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2003 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com DESIGN AND CHARACTERIZATION OF FUNCTIONAL NOVEL OLIGOPEPTIDES ONG BOON TEE (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOE THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2003 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com ACKNOWLEDGEMENT I would like to acknowledge Dr. Suresh Valiyaveettil for his guidance and advice throughout my Master’s research work. I would like to give my warmest gratitude to the following laboratory technicians: Ms Tang Chui Ngoh for her help with the SEM machine, Ms Kho Say Tin from the Department of Biological Sciences for her help with the HPLC and ESI-MS machine. A special thank to Assoc. Prof. Xu Guo Qin in allowing me to use the AFM machine in his laboratory. Next, I would like to thank my personal friend, Ms Michelle Low Bee Jin, in her help with the instruments when it’s faulty and also her encouragement and support during the duration of my Master’s research program. A special thanks to the following post-docs, Dr. Parayil Kumaran Ajikumar and Dr. Lakshminarayanan Rajamani, in their helpful and invaluable advice and encouragement during the course of my research. I would also like to extend my gratitude to the other postgraduate students and postdoctoral fellows in the group whom have in one way or another contributed their knowledge and help in the course of my research. Finally, I like to thank my family members for being there for me when I needed them most for their support and encouragement. PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com TABLE OF CONTENTS Acknowledgement Table of Contents i List of Abbreviations iv List of Tables vi List of Schemes vi List of Figures vii Summary x Chapter 1: Introduction 1 1.1 Introduction 2 1.2 Self-Assembly 2 1.3 Biomineralization 5 1.4 Outline of Thesis 1.5 1.4.1 Aim and scope of present work 12 References 13 Chapter 2: Synthesis and Characterization of Self-Assembly Peptides 18 2.1 19 Materials and Methods -iPDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 2.2 Solid-Phase Peptide Synthesis 19 2.3 Purification and Characterization of Peptides 23 2.4 Self-Assembly of Peptides 25 2.4.1 Dynamic Light Scattering (DLS) 25 2.4.2 Circular Dichroism (CD) Experiments 26 2.4.3 Atomic Force Microscopy (AFM) 27 2.5 Calcium Carbonate Crystallization Assay 27 2.6 Energy-Dispersive X-ray Scattering (EDXS) 30 2.7 Powder X-ray Diffraction (XRD) 30 2.8 References 30 Chapter 3: Results and Discussion 32 3.1 Introduction 33 3.2 Synthesis, Purification and Characterization of Peptides 34 3.3 Solution and Solid-state Structures of Synthetic Peptides (P1-P4) 36 3.3.1 Dynamic Light Scattering (DLS) studies 36 3.3.2 Circular Dichroism (CD) studies 41 3.3.2.1 In water 41 3.3.2.2 In 10 mM CaCl2 solution 43 3.3.2.3 Effect of salt solutions on solution conformations of 3.3.2.4 the peptides 45 pH dependent 47 - ii PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 3.4 Atomic Force Microscopy Studies of the peptides (P1 – P4) 49 3.4.1 In water at high concentration (1 mg/ml) of the peptides at pH ∼ 3 and 5 3.4.2 Influence of salt solutions on the self-assembly of peptides 3.5 3.6 49 57 Effects of the calcite crystals morphologies in the presence of peptides 3.5.1 Scanning Electron Microscopy (SEM) 64 3.5.2 Energy Dispersive X-Ray Scattering (EDXS) 72 3.5.3 Powder X-Ray Diffraction (XRD) 73 References 74 Chapter 4: Conclusions 77 4.1 Conclusion 78 4.2 References 80 - iii PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com List of Abbreviations A 0.1 % TFA in water ACN acetonitrile AFM atomic force microscopy Ala (A) alanine Arg (R) arginine Asp (D) aspartic acid B 0.1 % TFA in 80 % acetonitrile CD circular dichroism DLS dynamic light scattering EDXS energy dispersive X-ray scattering ESI-MS Electrospray Ionisation Mass Spectroscopy Fmoc 9-Fluorenylmethoxycarbonyl Glu (E) glutamic acid Gly (G) glycine h hours HATU 2-(1H-9-Azabenzotriazole-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate Ile (I) isoleucine Leu (L) leucine Lys (K) lysine µg micro gram - iv PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com µL micro litre mdeg milli degrees mg milli gram mL milli litre nm nanometer PAL 5-(4-Aminomethyl-3,5-dimethoxyphenoxy)valeryl Phe (F) phenylalanine pI isoelectric point Proline (P) proline RP-HPLC reversed phase high-pressure liquid chromatography SEM scanning electron microscopy SPPS solid-phase peptide synthesis TFA trifluoroacetic acid TIPS triisopropylsilane UV-CD ultraviolet circular dichroism XRD X-ray diffraction -vPDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com List of Tables Chapter 3 Table 1 Amino acid sequence, theoretical and observed masses and 35 percentage yield of the synthetic peptides Table 2 Quantitative analysis of the peptides at 1 mg/mL in water using 42 CDNN software Table 3 Quantitative analysis of the peptides at 2 mg/mL in 10 mM 44 CaCl2 solution using CDNN software Table 4 Quantitative analysis of the peptides at 1 mg/mL in 10mM 46 NaCl and CaCl2 solution respectively Table 5 Quantitative analysis of the peptides at 1 mg/mL in water at 48 pH ∼ 3 and 5 List of Schemes Chapter 2 Scheme 1 Mechanism involved in the formation of the calcium carbonate 29 crystals - vi PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com List of Figures Chapter 2 Figure 1 General scheme for Fmoc chemistry 22 Figure 2 Experimental set-up for calcite crystallization in the presence 29 of peptides Chapter 3 Figure 3 Purification of the peptides using RP-HPLC 35 Figure 4 Particle size distributions of the peptides (P1 to P4) in water 37 obtained by DLS at pH ~ 3 and 5 Figure 5 Particle size distributions of the peptides (P1 to P4) in 10 mM 39 CaCl2 and NaCl solutions obtained by DLS Figure 6 CD spectra of the peptides (P1 to P4) in water at various 41 concentrations Figure 7 CD spectra of the peptides (P1 to P4) in 10 mM CaCl2 solution 43 at various concentrations Figure 8 CD spectra of the peptides (P1 to P4) at 1 mg/ml in 10 mM 45 NaCl and CaCl2 solution respectively Figure 9 CD spectra of the peptides (P1 to P4) at 1 mg/ml in water at 47 pH ~ 3 and 5 at 25 °C - vii PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Figure 10 AFM images of P1 adsorbed onto mica substrate from water 49 at pH ~ 3 and 5 Figure 11 AFM images of P2 adsorbed onto mica substrate from water 51 at pH ~ 3 and 5 Figure 12 AFM images of P3 adsorbed onto mica substrate from water 53 at pH ~ 3 and 5 Figure 13 AFM images of P4 adsorbed onto mica substrate from water 55 at pH ~ 3 and 5 Figure 14 AFM images of P1 adsorbed onto mica substrate from 10 mM 57 CaCl2 and NaCl salt solutions Figure 15 AFM images of P2 adsorbed onto mica substrate from 10 mM 58 CaCl2 and NaCl salt solutions Figure 16 AFM images of P3 adsorbed onto mica substrate from 10 mM 60 CaCl2 and NaCl salt solutions Figure 17 AFM images of P4 adsorbed onto mica substrate from 10 mM 61 CaCl2 and NaCl salt solutions Figure 18 Two possible mechanisms for surface adsorption of 63 macromolecules in aqueous media. Figure 19 SEM micrographs of calcite crystals in the presence of P1 at 65 various concentrations Figure 20 SEM micrographs of calcite crystals in the presence of P2 at 67 various concentrations Figure 21 SEM micrographs of calcite crystals in the presence of P3 at 68 - viii PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com various concentrations Figure 22 SEM micrographs of calcite crystals in the presence of P4 at 70 various concentrations Figure 23 EDXS spectra of the crystal surface of the four peptides (P1 to 72 P4) at 2 mg/mL Figure 24 XRD of single crystals formed at 2 mg/mL of the four peptides 73 (P1 to P4) - ix PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Summary Molecular self-assembly is a unique and powerful method for assembling building blocks for functional materials and devices. Self-assembly of nucleic acid and protein are particularly interesting due to the tremendous approaches in the modern life sciences and material sciences. Most of these water-based systems are biocompatible, biodegradable and responsive to moderate changes in the media properties (like pH, temperature, ionic composition, etc.). Many groups have tried to understand the self-assembly of natural proteins by designing new oligomeric peptide chains, which form either solid crystals of well-defined architecture, nanotubes, or macroscopic membranes. Towards this direction we designed and investigated the self-assembly of a few novel peptides at different conditions. Herein we report the design strategy, synthesis and characterization of four peptides and their self-assemblies in different environments and the role in the crystallization of CaCO3. Two areas were studied in this research: (1) self-assembly of the peptides and (2) understanding of the protein-mineral interaction through biomineralization. In the first part of the work, the DLS results showed that the particle size distributions for all the peptides increased as the pH increase from 3 to 5. The same trend is observed in the salt solutions; the peptides in NaCl solution have a larger particle size distribution than in CaCl2 solution. In the CD spectra, all peptides except P4 gave a random coil conformation with some degree of a bend/β-turn conformation, whereas P4 gave a βsheet structure. From the AFM images, it was found that peptide 3 (P3) and peptide 4 (P4) gave fiber-like structures on a mica substrate, whereas spherical particles were -xPDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com observed for the peptides (P1 and P2). One reason for this finding is that both P3 and P4 gave an overall neutral charge, whereas P1 and P2 have an overall negative charge, which implies that, peptides with an overall neutral charge have the ability to form fiber-like structure. In the last section of the thesis, the role of the peptides on the nucleation of CaCO3 crystals was studied. All peptides did not induce any aggregation or polymorph nucleation, except that “hopper” crystals were formed due to non-specific binding. - xi PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com CHAPTER 1 INTRODUCTION -1PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com CHAPTER 1: INTRODUCTION 1.1 Introduction Molecular self-assembly has emerged as a new approach in chemical synthesis, nanotechnology, polymer science, material science and engineering. The preparation of materials via molecular self-assembly allows one to define material properties by the careful design of individual constituent molecules. Self-assembling systems investigated so far involve bi- and tri-block copolymers, small molecules, proteins and peptides. Using peptides as the molecular building blocks for self-assembly offers the possibility of incorporating biofunctionality into the material. 1.2 Self-Assembly of Peptides Molecular self-assembly is the spontaneous organization of molecules under thermodynamic equilibrium conditions to form structurally well-defined and stable arrangements through non-covalent interactions and is ubiquitous in nature at both macroscopic and microscopic scales [1-3]. The key engineering principle for molecular self-assembly is to artfully design the molecular building blocks that are able to undergo spontaneous assembly through the formations of numerous non-covalent weak interactions. These typically include hydrogen bonds, ionic bonds and van der Waals’ forces to facilitate the assembly of molecules into well-defined and stable hierarchical macroscopic structures [4]. Although individual non-covalent bonds are rather weak, the collective interactions can result in very stable structures. The key elements in molecular -2PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com self-assembly are chemical and structural complementarity. Like hands and gloves, both the size and the correct orientation, i.e. chirality, are important in order to have a complementary and compatible organization. Molecular self-assembly in nature Biomimicry and designing nature-inspired materials through molecular self-assembly is an emerging field of research in recent years. Nature is a grand master at designing chemically complementary and structurally compatible constituents for molecular selfassembly through eons of molecular selection and evolution. Chemical evolution from the first groups of primitive molecules through countless iterations of molecular selfassembly and disassembly has ultimately produced more and more complex molecular systems. In the last decade, considerable advances have been made in the use of peptides, phospholipids and DNA as building blocks to produce potential biological materials for a wide range of applications [5-13]. The constituents of biological origins, such as phospholipid molecules, amino acids and nucleotides have been considered to be useful building blocks for traditional materials science and engineering. The advent of biotechnology and genetic engineering coupled with the recent advancement in chemistry of nucleic acids and peptide syntheses has resulted a conceptual change in this area. Molecular self-assembly is emerging as a new route to produce novel materials that can complement the conventional synthesis of materials such as ceramics, metals and alloys, synthetic polymers and other composite materials. Several recent discoveries and rapid -3PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com developments in biotechnology have rekindled the field of biological materials engineering [14-16]. There are ample examples of molecular self-assembly in nature. One of the well-known examples is silk. The monomeric silk fibroin protein is approximately 1 mm but a single silkworm can spin fibroins into silk materials over 2 km in length, two billion times longer [17-18]! Such engineering skills can only make us envy of this biomacromolecule. Human ingenuity and current advances in technology is far behind the seemingly easy task achieved by the silkworm or spider. These building blocks are often at the nanoscale, however, the resulting materials could be measured at meters and kilometer scales. Likewise, the size of individual phospholipid molecules is approximately 2.5 nm in length, but they can self-assemble into millimeter-size lipid tubules with defined helical twist, many million times larger. A number of applications have been developed both for basic research and for potential applications in areas ranging from controlled release to electroactive composites [19]. Molecular self-assembly can also build sophisticated structures and materials. For example, collagen and keratin can self-assemble into macroscopic architectures such as ligaments and hair respectively. In cells, many individual chaperone proteins assemble into well-defined ring structures to sort out, fold and refold proteins [20]. The same is also true for other protein systems, involved in biomineralization processes responsible for the formation of hard tissues such as bones, teeth and seashells [21]. -4PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 1.3 Biomineralization Biomineralization is the selective and controlled production of organic-inorganic composite materials by living organisms [22-41]. It has occurred for millions of years. Half of these biogenic minerals contain calcium; for example, the teeth of the sea urchin contain calcium carbonate (CaCO3) in the form of calcite (the most common polymorph existing in nature), and primitive mollusks have aragonite spicules. In humans, biomineralization is observed not only during skeletal formation, but also in biological fluids generally supersaturated with a calcium salt, such as oxalate in urine, phosphate in saliva, or carbonate in pancreatic juice. This results in the formation of calcium salt crystals. While beneficial for tooth or bone mineralization, precipitation of calcium salts can be extremely harmful in fluids because it leads to the formation of stones and to the development of a lithiasis. A variety of minerals such as calcium carbonate, hydroxyapatite, silicate, and iron oxides are employed as biominerals. The control of the crystal shape/morphology of calcium carbonate is important for its industrial uses as pigments, fillers, dentifrices and of its biological role as structural supports in skeletons [42-45]. Calcium carbonate is the most abundant mineral observed in nature and exists in three forms, namely calcite, vaterite and aragonite [46]. Among these, calcite is the most thermodynamically stable form and vaterite is the least stable. Every organism has adapted certain strategic principles to optimize the specific function of its hard tissue to the specific environment in which it lives. Analysis of a variety of mineralizing biosystems leads to the following general principles that have significant implications for both biology and materials science: -5PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 1) Biomineralization occurs within specific subunit compartments or microenvironments, which implies stimulation of crystal production at certain functional sites and inhibition of the process at other sites; 2) A specific mineral is produced with a defined crystal size and orientation; or 3) Macroscopic growth is accomplished by the incremental growth of unique biocomposites. The effectiveness of the crystal growth and inhibition processes depends on the structure and chemistry of the interfaces between organic substrate, mineral, and medium. The highly specific control of morphology, location, orientation, and crystallographic phase all indicate the existence of an optimized or “engineered” substrate surface. The key characteristics of these optimized interfaces are elusive at present because of the complexity of most biological model systems. However, investigations of the representative systems, such as narce, dentin, enamel, cartilage, bone and avian eggshells, suggest a few basic principles of the biomineralization process. The following summarizes the key sequence of events known to operate in biomineralization processes and highlights the importance of coupled dynamics, microenvironments, and orientation between the organic matrix and the inorganic precursors [47]. -6PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Strategic elements of biomineralization Biomineralization occurs within specific subunit compartments [47]. 1) The dimensions of the compartment are established by the spatial distribution of a cell-derived biopolymer matrix, which self-assembles into arrays of oriented fibers or sheets and incorporates intrinsic domains that control the crystal formation process. 2) Outside of the “active” compartment, mineralization is actively inhibited by a variety of molecular processes. 3) The process of crystal nucleation and growth are separated temporally and regulated by complementary and redundant feedback control loops, which are crucial for countering the thermodynamic driving force leading to unrestricted mineralization from a supersaturated environment. 4) Nucleation of minerals within the matrix is actively controlled at the macromolecular level by specific initiation domains-genetically directed initiation steps are required for normal mineral development. 5) Supersaturation of the compartment is effected by any of a potentially wide array of ion delivery vehicles or pumps, which currently are poorly understood. These may include one or more of the following: (i) microencapsulated ions (matrix vesicles); (ii) polyelectrolytes; (iii) phosphoproteins or other Ca2+-binding proteins; (iv) phospholipids; and (v) enzyme catalysts to liberate nascent ions. 6) The density of the developing biomineral may be increased by removing organic templates or protecting groups or both – these regions may be backfilled with additional inorganic crystal at a later time. -7PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com A specific mineral is produced with defined crystal size and orientation [47]. 1) Because of the matrix architecture and chemistry, a specific crystal habit is achieved and its growth is highly directional relative to the organic phase. 2) Crystal selectivity is often accomplished by tailored initiation sites that may include: (i) periodic, negatively charged surfaces; (ii) bifunctional scaffolding molecules, and (iii) epitaxial elements containing a critical number of sites for nucleation. 3) Most of the crystals grow within the matrix structure. 4) Some matrix molecules may be incorporated within the crystal lattice. 5) In some cases, the mineral phase can be resorbed or remodeled, generally by cellmediated processes different from the original mineralization steps. Macroscopic growth is accomplished by packaging many incremental units together [47]. 1) Matrix-generating cells create a compartment (unit) or single layer forming one side of compartments. 2) Each compartment is processed to full density and shape. 3) The compartment secretion process is repeated for the next unit or layer of units, thereby producing a “moving front” of mineral deposition. 4) In most cases (for example, bone and nacre), biomineralization occurs very slowly, forming thin crystals or matrix lamellae perpendicular to the direction of growth. When rapid biomineralization occurs (avian eggshell), columnar crystals surrounded by matrix formed parallel to the direction of growth. -8PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Many living organisms contain biominerals and composites with finely tuned properties, reflecting a remarkable level of control over the nucleation, growth and shape of the constituent crystals. The formation of biominerals is controlled by organic template molecules resulting in materials with unique shapes and properties. In general, many different types of soluble additives, such as ions, organic molecules, macromolecules and polymers, present in the crystallization solution can block the incorporation of mineral ions into the crystal surface through adsorption at the kink and step sites. This interference gives rise to the inhibition of crystal growth or changes in the properties and morphology of the crystal. For example, it is well known that at high concentrations, Mg2+ ions influence the polymorph selectivity in CaCO3 crystallization [48]. This precipitation is due to the kinetic effects arising from the interaction of Mg2+ ions with small crystals and nuclei of calcite phase, which disrupts the surface and reduces the rate of crystal growth. At the same time, aragonite nuclei, which are not affected by the additive, continue to grow unabated in the supersaturated solution and therefore become the dominant polymorph in the crystallization process [49]. Peptides and proteins play an important role in achieving this polymorph selectivity. Peptides are useful analogues of proteins and have been used extensively to probe the role of functional motifs in altering the kinetics of crystal growth processes [50-53]. Based on the partial amino acid sequence available from the mollusk shells nacre, Levi et al. synthesized a series of peptides containing hydrophobic and hydrophilic amino acids and found that the peptides with stretches of poly(Asp-Leu) domains induced aragonite nucleation when adsorbed onto the chitin-silk fibroin complex [54]. Based on the -9PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com structure of type 1 anti-freeze protein from winter flounder, DeOlebra et al. synthesized peptide that contains stretches of aspartic acid and showed that the designed peptide binds to the {1 –1 0} calcite face [55]. Recently peptides derived from phage display have been studied for their role in metal binding [56], crystal nucleation [57], and structure-function relationships [58]. Thus peptides containing lesser number of amino acids are useful templates for understanding the biomineralization process. Strategies in Solid-Phase Peptide Synthesis (SPPS) Common elements in any chemical synthesis of peptides or proteins are the assembly of protected amino acids or peptide chains, their deprotection, purification and characterization. The basic strategy of SPPS still persists as the initial idea, outlined by Merrifield [59]. The process requires a solid support to attach the first amino acid residue and subsequent stages of the peptide as it is lengthened. The carboxyl end of the peptide is attached to the polymeric support. The N-terminus needs protection and deprotection at each stage of the stepwise synthesis and which results amino group after each deprotection. The N-protected amino acid is activated for coupling to the growing peptide chain. For stepwise elongation of peptides on a polymeric support, these three steps, deprotection, neutralization and coupling would be repeated until the desired sequence is assembled. Finally the covalent bond to the solid support is cleaved to obtain the free peptide. The potential advantages of this proposed synthetic strategy are: - 10 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com • Speed, simplicity and high yield. • Solid support with peptides was washed by simple filtration without transfer to other containers, which avoids physical losses. • All the chemical reactions during synthesis, deprotection, neutralization and coupling reactions would be driven to completion by an excess and high concentration of soluble reagents. • It would be possible to efficiently remove excess reagents and soluble byproducts by washing with large excess of solvents, to effect a rapid partial purification after each step. • A complete automation of the entire synthesis is possible. At the same time, some of the potential disadvantages of the stepwise SPPS involve incomplete reactions and the gradual buildup of insoluble by-products [60]. - 11 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 1.4 Outline of Thesis 1.4.1 Aim and scope of present work Here in this present work, we designed and synthesized peptides which are expected to form stable secondary structures and interesting materials. The fundamental which leads to the designing of these peptides emerged from the fact that peptides with alternating hydrophobic-hydrophilic residues are known to self-assemble into β-sheet structures, often in an ionic-strength-dependent manner [61-62]. pH or salt-induced self-assembly of such peptides may be driven by the shielding of electrostatic repulsive forces with increasing ion concentration, allowing attractive hydrophobic and van der Waals forces to dominate [63]. Three of the peptides synthesized consist of alternating hydrophobic and hydrophilic residues. Out of these three peptides, two of them consist of a cell adhesion motif ‘RGD’ in the middle of the peptide. This tripeptide motif is a well studied and an important ligand for some members of the integrin family of the cell adhesion receptors. The fourth peptide is incorporated with a mimic of the cell adhesion motif ‘RAD’. To determine whether only the above design will give stable secondary structures, another peptide was designed and synthesized which consist of mostly hydrophobic amino acid residues with a mimic of the cell adhesion motif ‘KGD’ situated in the middle of the peptide. The purpose of incorporating these biologically active motifs into the amino acid sequence is to determine whether these motifs have any influence in the self-assembly properties. Herein we investigated the role of the designer peptides in the self assembly at different pH or salt solutions. - 12 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com In this work, four peptides were synthesized and their self-assembly in aqueous solutions at different pH and in the presence of metal ions to obtain valuable information on secondary structures. Chapter 2 presents the experimental section, which gives a detailed description of the experimental procedures employed for the synthesis, purification and characterization of the four peptides. 1.5 References 1. Whitesides, G. M.; Mathias, J. P.; Seto, C. T. Science 1991, 254, 1312-1319. 2. Lehn, J. M. Science 1993, 260, 1762-1763. 3. Ball, P. Nature 1994, 367, 323-324. 4. Pauling, L. Nature of the Chemical Bond and the Molecular Crystals: an Introduction to Model Chemistry; 3rd edn., Cornell University, Ithaca, NY, 1960. 5. Schnur, J. M. Science 1993, 262, 1669-1676. 6. Ghadiri, M. R.; Granja, J. 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Ed. Engl. 1992, 31, 153-169. 26. Mann, S. Nature 1988, 332, 119-124. - 14 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 27. Weiner, S.; Addadi, L. J. Mater. Chem. 1997, 7, 689-702. 28. Calvert, P.; Mann, S. J. Mater. Sci. 1988, 23, 3801-3815. 29. Mann, S. in Inorganic Materials; 2nd ed. (Eds: D.W. Bruce, D. O’Hare); Wiley, Chichester, UK, 1996, 255. 30. Biominerlization (Ed: E. Baeuerlein); Wiley-VCH, Weinheim, 2000. 31. Watabe, N. Biomineraliztion (in Japanese); Tokai University Press, Tokyo, 1997. 32. Krampitz, G.; Graser, G. Angew. Chem. Int. Ed. Engl. 1988, 27, 1145-1156. 33. Addadi, L.; Weiner, S. Nature 1997, 389, 912-915. 34. Sikes, C. S.; Wheeler, A. P. CHEMTECH 1988, 620-626. 35. Smith, B. L. Chem. Ind. (London) 1998, 16, 649-653. 36. Mann, S.; Ozin, G. A. Nature 1996, 382, 313-318. 37. Aksay, I. A.; Trau, M.; Manne, S.; Honma, I.; Yao, N.; Zhou, L.; Fenter, P. M. E.; Gruner, S. M. Science 1996, 273, 892-898. 38. Stupp, S. I.; Braun, P. V. Science 1997, 277, 1242-1248. 39. Kato, T. Adv. Mater. 2000, 12, 1543-1546. 40. Watabe, N. J. Ultrastruc. Res. 1965, 12, 351-363. 41. Nakahara, H.; Bevelander, G.; Kakei, M. Venus Jpn. J. Malacol., 1982, 41, 33-40. 42. Schäffer, T. E.; Ionescu-Zanetti, C.; Proksch, R.; Fritz, M.; Walters, D. A.; Almqvist, N.; Zaremba, C. M.; Belcher, A. M.; Smith, B. L.; Stuchy, G. D.; Morse, D. E.; Hansma, P. K. Chem. Mater. 1997, 9, 1731-1740. 43. Fendler, J. H.; Meldrum, F. C. Adv. Mater. 1995, 7, 607-632. 44. Berman, A.; Hason, J.; Leiserowitz, L.; Koetzle, T. F.; Weiner, S.; Addadi, L., Science 1993, 259, 776-779. - 15 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 45. Heywood, B. R.; Mann, S. Adv. Mater. 1994, 6, 9-20. 46. McGrath, K. M. Adv. Mater. 2001, 13, 989-992. 47. Heuer, A. H.; Fink, D. J.; Laraia, V. J.; Arias, J. L.; Calvert, P. D.; Kendall, K.; Messing, G. L.; Blackwell, J.; Rieke, P. C.; Thompson, D. H.; Wheeler, A. P.; Veis, A.; Caplan, A. I. Science 1992, 255, 1098-1105. 48. Raz, S.; Weiner, S.; Addadi, L. Adv. Materials 2000, 12, 38-42. 49. Loste, E.; Wilson, R. M.; Seshadri, R.; Meldrum, F.C. J. of Crystal Growth 2003, 254, 206-218. 50. Mann, S. J. Mater. Chem. 1995, 5, 935-946. 51. Oates, J. A. H. Lime and Limestone: Chemistry and Technology, Production and Uses; Wiley-VCH, Weinheim, 1998. 52. Levi, Y.; Albeck, S.; Brack, A.; Weiner, S.; Addadi, L. Chem. Eur. J. 1998, 4, 389-396. 53. DeOliveira, D. B.; Laursen, R. A. J. Am. Chem. Soc. 1997, 119, 10627-10631. 54. Wustman, B. A.; Morse, D. E.; Evans, J. S. Langmuir 2002, 18, 9901- 9906. 55. Wustman, B. A.; Santos, R.; Zhang, B.; Evans, J. S. Biopolymers 2002, 65, 362372. 56. Zhang, B.; Wustman, B. A.; Morse, D. E.; Evans, J. S. Biopolymers 2002, 63, 358- 369. 57. Zhang, B.; Xu, G. Z.; Evans, J. S. Biopolymers 2000, 54, 464- 475. 58. Xu, G. Z.; Evans, J. S. Biopolymers 1999, 49, 303-312. 59. Merrifield, R. B. In Fed. Proc. Amer. Soc. Exp. Biol. 1962, 21, 412-428. - 16 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 60. Ajikumar, P. K. PhD Thesis; School of Chemical Sciences, Mahatma Gandhi University, India, 2001. 61. Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3334-3338. 62. Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Biopolymers 1994, 34, 663-672. 63. Caplan, M. R.; Schwartzfarb, E. M.; Zhang, S.; Kamm, R. D.; Lauffenburger, D. A. Biomaterials 2002, 23, 219-227. - 17 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com CHAPTER 2 SYNTHESIS, PURIFICATION AND CHARACTERIZATION - 18 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com CHAPTER 2: SYNTHESIS, PURIFICATION AND CHARACTERIZATION 2.1 Materials and Methods Fmoc-Ala-PEG-PS, Fmoc-Ile-PEG-PS and all the Nα-Fmoc-L-amino acids were purchased from Novabiochem, triisopropylsilane(TIPS), San Diego dimethylformamide CA. (DMF, azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium Trifluoroacetic synthesis acid grade), hexafluorophosphate (TFA), 2-(1H-9(HATU), piperidine solution and diisopropylethylamine (DIPEA) were purchased from Applied Biosystems and used without further distillation unless otherwise stated. Anhydrous diethyl ether (reagent grade), methanol (HPLC grade) and acetonitrile (HPLC grade) were used as received. Pure calcium chloride dihydrate (CaCl2.2H2O) and ammonium bicarbonate ((NH4HCO3) were used as received. Millipore water was used to prepare the buffers for the HPLC purification and other characterizations. 2.2 Solid-phase peptide synthesis In 1984 Bruce Merrifield, an American chemist at Rockerfeller University won the Nobel Prize for his contribution to the advancement of peptide chemistry. He developed a solidphase peptide synthesis (SPPS) methodology of peptides, which uses a polymer with reactive sites (solid supports) that allow the addition of amino acid residues stepwise to synthesize peptide chains using a stepwise mechanism [1]. In the Merrifield’s technique, - 19 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com the problems associated with low yields due to separation and purification is avoided. The insoluble polymer can be filtered and washed without losses [1]. Solid-phase peptide synthesis consists of three distinct sets of operations: (1) chain assembly of peptide chain on a resin; (2) simultaneous or sequential cleavage and deprotection of the resin-bound, fully protected peptide chain; and (3) purification and characterization of the target peptide. Various chemical strategies exist for the chain assembly and cleavage/deprotection operations, but purification and characterization methods are more or less invariant to the methods used to generate the crude peptide product. The acid-labile “Boc” group or base-labile “Fmoc”-group is used for N-α-protection [2]. After removal of this protecting group, the next protected amino acid is added using either a coupling reagent or pre-activated protected amino acid derivative. The resulting peptide is attached to the resin, via a linker, through its C-terminus and may be cleaved to yield the desired peptide. Side-chain protecting groups are often chosen so as to be cleaved simultaneously with detachment of the peptide from the resin. Cleavage of the Boc protecting group is achieved using trifluoroacetic acid (TFA) and the Fmoc protecting group using piperidine solution [2]. Final cleavage of the peptidyl resin and side-chain deprotection requires strong acid, such as hydrogen fluoride (HF) or trifluoromethanesulfonic acid (TFMSA), in the case of Boc chemistry, and TFA in Fmoc - 20 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com chemistry. Dichloromethane (DCM) and N,N-dimethylformamide (DMF) are the primary solvents used for resin deprotection, coupling and washing. Peptide synthesis can be carried out in a batch-wise or continuous flow manner. In the former technique, the peptidyl resin is contained in a filter reaction vessel and reagents added and removed under manual or computer control. In the continuous flow method, the resin is contained in a column through which reagents and solvents are pumped and removed continuously. A range of manual, semi-automatic or automatic synthesizers are commercially available for both batch-wise or continuous flow methods. Only the Fmoc strategy is fully compatible with the continuous flow method in which real-time spectrophotometric monitoring of the progress of coupling and deprotection is also possible. In this study, the oligopeptides were synthesized using the Pioneer peptide synthesizer from the Applied Biosystems. The solid-state peptide synthesis principle using the Fmoc chemistry was used. The general procedure is given below: - 21 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com R R Fmoc OH Attachment + HO Fmoc O N H N H O O O R R Fmoc H N O N H Fmoc Deprotection N H O R Activating Group O O H2N Coupling R O O Repeat Deprotection and Coupling O R H N O Cleavage and Deprotection Peptide and Polymer N H Fmoc R n O Figure 1. General scheme for Fmoc chemistry Peptides are usually purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using columns such as C18, C8 or C4 depending on the molecular weights of the polypeptides. The first Fmoc amino acid was attached to an insoluble support resin via an acid labile linker. Deprotection of the Fmoc, is accomplished by treatment of the resin with a base, usually piperidine. The second Fmoc amino acid was coupled utilizing a preactivated species or in situ activation. The coupling agent used for all synthesis was HATU. After the desired peptide was synthesized, the peptide was deprotected and detached from the solid support via TFA cleavage. - 22 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com The completed peptides were deprotected and cleaved by treating with either cleavage cocktail R (90% trifluoroacetic acid (TFA), 2.5% phenol, 2.5% water, 2.5% thioanisole, 2.5% ethanedithiol) or cleavage cocktail B (88% TFA, 5% phenol, 5% water, 2% TIPS) for 3-5 hours depending on the sequences. All the deprotection and cleavage were carried out at room temperature. The mixture was filtered and concentrated to reduce the volume of the filtrate. The peptide was precipitated by adding ice-cold diethyl ether. For maximum recovery, the precipitated peptide together with the ether layer was put in the freezer overnight. This mixture was centrifuged using an ultracentrifuge with repeated washing by ice-cold ether to remove all contaminating agents. Finally the peptides were lyophilized with 10% acetic acid solution and obtained as white powder. 2.3 Purification and characterization of peptides By far the most common technique for the purification of peptides is RP-HPLC. This is a very powerful method that allows the separation of peptides from a variety of impurities, including side products in which one of the amino acids has undergone partial racemization. RP-HPLC is an excellent technique to separate the proteins based on hydrophobicity of the samples. It requires the optimization of parameters such as choice of the column, slope of the eluting gradient and pH of the buffers. RP-HPLC may not be useful for large and hydrophobic proteins owing to prior elution or require high concentration of the organic solvent. Less hydrophobic column could be used instead of a more conventional C8 or C18 columns. In this work, a C18 column was used for the final purification process. The highly non-polar surface in a C18 column preferentially interacts - 23 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com with the hydrophobic molecules (such as most peptides) if they are introduced in a polar mobile phase. Electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) have found widespread utility for characterization of the peptides. Here, we used the ESI-MS for the analysis of the four peptides synthesized. In ESI-MS, peptide fragmentation can be obtained through collisionally activated decomposition (CAD, also called collision-induced dissociation, CID). Analysis of the CAD products is most efficiently accomplished by a multistage instrument, such as a triple quadrupole, a double-focusing magnetic sector instrument, or an ion trap, after mass selection of the ion of interest. The crude peptides were fractionated on a Jupiter C18 reversed phase column (5μ, 250 mm x 10 mm) using a Vision Workstation (Perkin - Elmer PerSeptive Biosystems). The solvent system used for the purification was: solvent A: 0.1% TFA in water and solvent B: 0.1% TFA in 80% acetonitrile. A linear gradient (2 mL/min) of 25%-50% B over 40 min was used. The column was equilibrated with 0.1% trifluoroacetic acid and a linear gradient of acetonitrile was used for elution. The crude peptides (~5 mg protein) were injected onto the column and were eluted at a flow rate of 2 mL/min. The elution of the peptides was monitored both at 215 and 280 nm. Precise masses of the peptides were determined by ESI-MS using a Perkin-Elmer Sciex API 300 triple quadrupole instrument equipped with an ion spray interface. The ion spray voltage was set at 4.6 kV and the orifice voltage at 30 V. Nitrogen was used as a curtain gas with a flow rate of 0.6 L/min while compressed air was utilized as the nebulizer gas. - 24 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com The sample was injected into the mass spectrometer at a flow rate of 50 µL/min and scanned from mass to charge (m/z) ratio of 500 to 2000. The multiply charged spectrum was deconvoluted into the mass scale using the Biospec Reconstruct software supplied with the instrument data system. 2.4 Self-assembly of peptides The self-assembly of ionic self-complementary oligopeptides was investigated by S. Zhang et. al [3]. These peptides are short, simple to design, extremely versatile, and easy to synthesize. Three types of self-assembling peptides have been systematically studied so far. This new class of biomimetic materials has considerable potential for a number of applications, including scaffolding for tissue repair and tissue engineering, delivery of molecular medicine, and biological surface engineering. Similar systems have also been described where these peptide systems undergo self-assembly to form a gel with regular β-sheet tapes of well-defined structure [4]. Furthermore, a number of fascinating biomimetic peptide and protein structures have been synthesized, such as helical coil-coil and di-, tri-, and tetrahelical bundles [5-7]. 2.4.1 Dynamic Light Scattering (DLS) The DLS studies were carried out with a 5-watt argon ion laser (Brookhaven Instruments) and SEM 615 nm gonimeter coupled to a real time correlator RTG of 12 channels. The - 25 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com power of the laser was varied from 50 to 500 mW depending upon the peptide concentration. The data were collected at a scattering angle of 90° to the incident laser beam and the supplying time was 0.8 µs. An aliquot of 150-200 µl of the peptide solutions (5 mg/mL) was used to perform DLS experiments by using PDDLS/batch light scattering instrument (Precision Detectors, Franklin, MA). Intensity data from each sample were collected in duplicate and analysed by using the PRECISION DECONVOLVE program and yielded size-versus-fraction distribution plots. 2.4.2 Circular Dichroism (CD) Experiments The secondary structure of the protein was analyzed using Jasco J 700 circular dichroism (CD) spectropolarimeter. The instrument was calibrated with water, 10 mM CaCl2 and 10 mM NaCl solutions respectively. The CD spectra of the peptides at a concentration range of 1 mg/mL - 125 µg/mL in water at room temperature were collected using 0.1 mm sample cell. To study the effect of Ca2+ ions, spectra were also recorded in 10 mM calcium chloride solution with a concentration range of 2 mg/mL – 50 µg/mL at room temperature. The CD spectra of the peptides (1 mg/mL) under different pH and salt solutions were also studied. The instrument optics was flushed with 30 L/min nitrogen gas. A total of three scans were recorded and averaged for each spectrum and baseline subtracted. The conformation of the peptides was analyzed using the CDNN software. - 26 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 2.4.3 Atomic Force Microscopy (AFM) AFM experiments were performed at room temperature in air with a Nanoscope IIIa scanning probe microscope equipped with an ‘E head’ scanner (Digital Instruments, Santa Barbara, CA). The peptides were each dissolved in water, to make a stock solution with a concentration of 1 mg/mL. Freshly cleaved mica were then immersed into 1 mL of the peptide solutions for about 15 mins to 1 hour, rinsed with water, the excess liquid was removed using filter paper, and dried at room temperature before the imaging was carried out. The images were acquired in the tapping mode using commercial cantilevers with sharpened silicon tips. 2.5 Calcium carbonate crystallization assay Although biochemical and structural properties of the proteins associated with various tissues have been well documented, only limited information is available on the spatial relations between these macromolecules and the mineral phase. Two basically different approaches were used. The first approach – the kinetic approach, evaluates the ability of the macromolecule to limit the nucleation process and to affect the rates at which the crystals grow [8]. By measuring change in the length of induction period prior to crystal formation, one would obtain information on whether the macromolecules inhibit or trigger the nucleation [9]. A wide variety of proteins and glycoproteins from mineralized tissues were evaluated using this approach [10-13]. The second approach, termed as “stereo chemical” approach, focuses on the manner in which the macromolecules - 27 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com influence the crystal growth through adsorption onto the crystal faces [14-15]. The key property monitored was the change in crystal morphology resulting from inhibition of growth of a particular crystal face onto which the peptide was adsorbed. The strength of this method lies in differentiating between specific and non-specific effects. Herein the second method was used to investigate the role of peptides in the biomineralization process. Calcium carbonate crystals were grown on glass cover slips placed inside the calcium chloride solution kept in a Nunc dish 4 x 6 wells. Typically, 1 mL of 7.5 mM calcium chloride solution was introduced into the wells containing the cover slips and the whole set up was covered with aluminium foil with a few pinholes on the top. To study the role of peptides in the calcium carbonate crystallization, aliquots of peptide dissolved in 7.5 mM calcium chloride solution at a concentration range of 50 µg/mL to 2 mg/mL were introduced into the crystallization wells containing glass cover slips. Crystals were grown inside a closed desiccator for 2 days by slow diffusion of CO2 released by the decomposition of ammonium bicarbonate (NH4HCO3) placed at the bottom of the desiccators. After 2 days, the glass slides were carefully lifted from the crystallization wells, rinsed gently with water, air dried at room temperature and used for characterization. The mechanism for the crystallization is given below: - 28 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com NH4HCO3 NH3 (g) + CO2 (g) + H2O HCO3- HCO3- NH3 CO32- + NH4 + Ca2+ 2Cl- CaCO3 (s) + NH4Cl Scheme 1 Mechanism involved in the formation of the calcium carbonate crystals desiccator RT CaCl2 solution and peptides NH4HCO3 (s) Cover glass-slip Figure 2 Experimental set-up for calcite crystallization in the presence of peptides. SEM studies on the calcium carbonate crystals were carried out using JEOL JSM-5200 scanning electron microscope at either 5 or 15 kV after coating the crystals with platinum to increase the conductivity. The coater used was JEOL JFC-1600 Auto Fine Coater. - 29 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 2.6 Energy-dispersive X-ray Scattering (EDXS) To determine the type of elements present in the calcium carbonate crystals, EDXS was carried out using Philips XL30 FEG. 2.7 Powder X-ray Diffraction (XRD) The crystals from the biomineralization experiments were analysed with XRD after characterizing the crystals with SEM. A V12 sample holder was used and the angles scanned were between 20 and 70 at a step size of 1°. The diffraction pattern was collected using Rigaku diffractometer with Cu-Kα radiation using Siemens D5005 X-Ray diffractometer at 40 kV and 40 mA. Each scan took about 45 mins. 2.8 References 1. Merrified, R. B. In Fed. Proc. Amer. Soc. Exp. Biol., 1962, 21, 412-428. 2. Atherton, E; Sheppart, R. C. Solid Phase Peptide Synthesis: A Practical Approach, Oxford: IRL Press, 1989, 25. 3. Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci., U.S.A. 1993, 90, 3334-3338. 4. Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.; McLeish, T. C. B.; Pitkeathly, M.; Radford, S. E. Nature 1997, 386, 259-262. 5. O’Shea, E. K.; Rutkowski, R.; Kim, P. S. Science 1989, 243, 538-542. - 30 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 6. Hecht, M. H.; Richardson, J. S.; Richardson, D. C. Science 1990, 249, 884-891. 7. Baker, D.; DeGrado, W. F. Curr. Opin. Struct. Biol. 1999, 9, 485-486. 8. Tracy, S. L.; Williams, D. A.; Jennings, H. M. J. of Crystal Growth 1998, 193, 382-388. 9. Nancollas, G. H.; Mohan, M. S. Arch. Oral. Biol. 1970, 15, 731-745. 10. Cuervo, L. A.; Pita, J. C.; Howell, D. S. Calcif. Tissue Res. 1973, 13, 1-10. 11. Termine, J. D.; Eanes, E. D.; Conn, K. M. Calcif. Tissue Res. 1980, 31, 247-251. 12. Wheeler, A. P.; George. J. W.; Evans, C. A. Science 1981, 212, 1397-1398. 13. Morneo, E. C.; Kresak, M.; Kane, J. J.; Hay, D. I. Langmuir 1987, 3, 511-519. 14. Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci., U.S.A. 1985, 82, 4110-4114. 15. Addadi, L.; Moradianoldak, J.; Shay, E.; Maroudas, N. G.; Weiner, S. Proc. Natl. Acad. Sci., U.S.A. 1987, 84, 2732-2736. - 31 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com CHAPTER 3 RESULTS AND DISCUSSIONS - 32 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com CHAPTER 3: RESULTS AND DISCUSSIONS 3.1 Introduction Molecular self-assembly has attracted considerable attention due to its use in the design and fabrication of nanostructures leading to the development of nanodevices [1-2]. There have been many elegant approaches to the design and fabrication of new self-assembling materials using oligopeptides into sheets, films and other structures [3]. There are many potential applications for self-assembled oligopeptides such as model systems for understanding the diseases like Alzheimer’s disease, scaffold in tissue engineering and controlled drug delivery [4]. Biomaterial scaffolds are used as components of cell-laden artificial tissues and transplantable biosensors. Some of the most promising new synthetic biomaterial scaffolds are developed from self-assembling peptides incorporated with cell surface recognition residues. Protein design ultimately comes down to choosing the appropriate amino acid sequences [5-6]. The global features important for designing novel proteins are obtained from the examination of a sequence of natural proteins. Such examination reveals two universal themes: (1) globular proteins fold into structures that maximize the shielding of hydrophobic side chains while simultaneously exposing the hydrophilic side chains to aqueous environment, (2) these structures typically form secondary structure such as αhelices and β-sheets. The prevalence of these two features among the natural proteins suggests that they play a crucial role in defining appropriate sequences that are most likely to yield soluble, well-folded de novo proteins. Sequences that are capable of - 33 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com forming regular secondary structure can be designed by selecting polar and nonpolar residues to match the structural periodicity of the desired secondary structure [7]. Towards this direction, we designed and synthesized four peptides, where amino acid residues were randomly chosen to determine the formation of stable secondary structures. The peptides were also used to investigate their self-assembly property and their ability to act as template for biomineralization. Two of them consist of alternating hydrophobic and hydrophilic residues with cell adhesion motif in the middle of the peptide. This tripeptide motif is a well studied and an important ligand for some members of integrin family of cell-adhesion receptors [8]. The third peptide consists of mainly hydrophobic amino acid residues with a mimic of the cell adhesion motif of “RGD” to “KGD”. Similar to P1 and P2, P4 consists of alternating hydrophobic and hydrophilic amino acid residues with repeating “RAD” moieties. 3.2 Synthesis, purification and characterizations of peptides The peptides were synthesized using a Pioneer peptide synthesizer (Applied Biosystems) using the Nα-Fmoc-L-amino acid/HATU coupling method on a 0.1 mmol scale using the extended cycle protocol. Finally the peptides were lyophilized with 10 % acetic acid solution and obtained as white powder. The crude peptides obtained after cleavage and lyophilization were purified on a BioCAD Workstation HPLC using the Phenominex C18 reverse-phase column (250 × 10 nm, 10 µm). Figure 3 shows the typical HPLC profiles of the different peptides over a gradient of 25-50 % B for 50 min. From the HPLC profile, peak at the highest intensity correspond to the desired peptide. The pure fractions - 34 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com were pooled, freeze dried and a white powder was obtained. The purity of the peptides Absorbance at 215 nm Absorbance at 215 nm was conferred by ESI-MS and the results were summarized in Table 1. Peptide 1 A 0 1000 2000 3000 Peptide 2 B 500 4000 1500 2500 Absorbance at 215 nm Absorbance at 215 nm Time (sec) Peptide 3 C 0 1000 2000 3500 4500 Time (sec) 3000 4000 Peptide 4 D 0 1000 2000 3000 4000 Time (sec) Time (sec) Figure 3. Purification of the peptides using RP-HPLC. (A) Peptide 1, P1; (B) Peptide 2, P2; (C) Peptide 3, P3 and (D) Peptide 4, P4. The bound peptides were eluted against a segmented gradient at a flow rate of 2 mL/min. Theoretical Observed mass † % Yield of mass (Da) (Da) the peptide ADAELDPRGDFPDLEADA, (P1) 1916.945 1916.53 ± 0.69 40.5 IELKPRGDFPDLEA, (P2) 1599.805 1599.72 ± 0.05 42.6 LPLLKGDLRI, (P3) 1137.455 1137.25 ± 0.69 55.8 RADARADARADARADA, (P4) 1671.695 1671.52 ± 0.74 60.4 Amino acid sequence Table 1. Amino acid sequence, theoretical and observed masses & percentage yield of the synthetic peptides. †: confirmed by ESI-MS. - 35 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 3.3 Solution and solid-state structures of the synthetic peptides (P1-P4) The secondary structure of the peptides in solution was investigated by circular dichroism (CD) and dynamic light scattering (DLS) in water, various salt solutions and different pH. The influence of the monovalent and divalent ions was investigated in the presence of 10 mM CaCl2 and 10 mM NaCl respectively. The ions were chosen because Na+ can only bind to one carboxylate group whereas Ca2+ interacts with two carboxylate groups. The effect of pH on the self-assembly of peptides was monitored at pH 3 and pH 5, and further studies are in progress. 3.3.1 Dynamic Light Scattering (DLS) For a more insight into the solution structures and the possibility of the supramolecular structure formation, we carried out the dynamic light scattering experiments of the peptidyl solutions (5 mg/mL) at different pH and salt solutions. - 36 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com A B C E D F G Figure 4. Particle size distributions of the peptides in water obtained by dynamic light scattering (DLS). (A) P1 at pH ∼ 3; (B) P1 at pH ∼ 5; (C) P2 at pH ∼ 3; (D) P2 at pH ∼ 5; - 37 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com (E) P3 at pH ∼ 5; (F) P4 at pH ∼ 3; (G) P4 at pH ∼ 5. X-axis indicates the size of the particles and Y-axis represents the fraction distribution. The dynamic light-scattering studies showed structures with very discrete sizes. As the pH increases from 3 to 5, there is an increase in the observed hydrodynamic radius () i.e. from 3.0 to 10.2 nm. Similarly in P2 the increases from 3.6 to 7.9 nm with a broadening of size distribution at pH ∼ 5 (Figure 4D). No DLS data was observed for P3 at pH ∼ 3, indicating that either no discrete nanostructures were formed or the particle sizes were too small to be detected and can no longer accurately measure the size of structures in the peptide solution of P3. The for P3 at pH ∼ 5 was 10.2 nm (Figure 4E), which shows the presence of nanostructures in the solution. This broadening in the size distribution may indicate that multimers were formed. The increase in values of the peptides at these two pH values indicated that pH induced formation of the aggregates in solution. The for the peptide P4 was found to be ∼ 6.3 nm at pH ∼ 3 (Figure 4F). It is important to note that all the peptides showed self-aggregation in solution. - 38 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com E A B C D F G Figure 5. Particle size distribution of the peptides in 10 mM CaCl2 and NaCl solutions obtained by dynamic light scattering (DLS). (A) P1 in CaCl2 ; (B) P1 in NaCl; (C) P2 in - 39 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com CaCl2; (D) P2 in NaCl; (E) P3 in NaCl; (F) P4 in CaCl2 ; (G) P4 in NaCl. X-axis indicates the size of the particles and Y-axis represents the fraction distribution. With salt solutions (Figure 5), the peptides formed smaller nanostructures compared to the pH induced self-assembly. In presence of NaCl (Figure 5B and 5D), P1 and P2 showed a broader distribution compared with the presence of CaCl2 solution (Figure 5A and C). P3 in NaCl solution (Figure 5E) showed highly monodisperse particles with ca. 2.1 nm, and the peptide P4 (Figure 5F and 5G) exists as multimers in NaCl and CaCl2 solutions. There was no discrete peak observed for P3 in CaCl2 solution may be due to the small particle size or the instrument was not sensitive enough to detect the particles. An increase in the was observed when the salt solution was changed from CaCl2 to NaCl. The reason for this trend might be due to the interaction of the cations with the carboxylate groups on the peptide. Na+ ions interact with the acid group with a 1:1 stiochiometry, whereas the Ca2+ forms a 2:2 complexes with the acid. This may be the reason for the Ca2+ complexes to have smaller size as compared to Na+ complexes. - 40 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 3.3.2 Circular Dichroism (CD) studies 3.3.2.1 In water -2 5 -12 -15 1 mg/ml CD (mdeg) CD (mdeg) -22 0.5 mg/ml -32 0.25 mg/ml 0.125 mg/ml -42 1 mg/ml -35 0.5 mg/ml 0.25 mg/ml 0.125 mg/ml -55 -52 -75 A -62 -72 190 200 210 220 230 240 250 B -95 190 260 200 210 Wavelength (nm) 220 230 240 250 260 Wavelength (nm) 10 5 0 -10 -5 -10 CD (mdeg) CD (mdeg) -30 1 mg/ml 0.500 mg/ml -50 0.250 mg/ml 0.125 mg/ml -70 1 mg/ml -15 0.5 mg/ml -20 0.25 mg/ml 0.125 mg/ml -25 -30 -90 C -110 D -35 -40 190 200 210 220 230 240 250 260 190 200 210 Wavelength (nm) 220 230 240 250 Wavelength (nm) Figure 6. CD spectra of the peptides in water at various concentrations. (A) P1; (B) P2; (C) P3; (D) P4. Figure 6 represents the CD spectra of the peptides studied at 25 °C in water at various concentrations. All the peptides gave a random coil conformation at around 198-199 nm - 41 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 260 [9] at low concentrations. As the concentration increases, no significant difference in the shape of the CD spectra was observed except that the intensity of the CD negative band increases from –71 mdeg to –105 mdeg for peptides P1 to P3. This shows that the conformation of these peptides is not sensitive to concentration changes which are in agreement with other oligopeptides of similar length [10-11]. The increase in intensity of the negative peak at increasing concentration indicates that P3 has a slightly more ordered structure compared to the other peptides (P1 and P2). There was a decrease in the intensity of peak on the P4 spectrum, which indicates that it has a random coil and αhelix conformation having a negative minimum of 199 nm and a positive maximum at 192 nm. A shoulder was also observed for peptides P1 to P3 between 228-230 nm indicating the presence of β-turn conformation. This is due to the incorporation of proline residue that is capable of introducing the turn-forming motif [12]. Quantitative analysis of the peptides at 1 mg/mL in water using the CD Spectra Devolution Software (CDNN) was summarized in Table 2. % Conformations for the peptides Peptides Conformation α-helix β-sheet β-turn Random coil P1 P2 P3 P4 7.0 36.0 21.5 35.5 11.1 35.6 23.2 32.0 7.5 34.7 24.9 33.7 6.1 42.6 19.1 33.0 Table 2. Quantitative analysis of the peptides at 1 mg/mL in water using CDNN software. - 42 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 3.3.2.2 In 10 mM CaCl2 solution 30 15 10 -5 CD (mdeg) CD (mdeg) 2 mg/ml -25 1 mg/ml 0.5 mg/ml 0.1 mg/ml -45 0.05 mg/ml -10 2 mg/ml 1 mg/ml 0.5 mg/ml -30 0.1 mg/ml 0.05 mg/ml -50 -65 -90 -105 190 200 210 220 230 240 250 B -70 A -85 190 260 200 210 220 230 240 250 260 Wavelength (nm) Wavelength (nm) 25 10 15 5 2 mg/ml CD (mdeg) CD (mdeg) -10 1 mg/ml 0.5 mg/ml -30 0.1 mg/ml 0.05 mg/ml -50 -5 2 mg/ml -15 1 mg/ml 0.5 mg/ml -25 0.1 mg/ml -35 0.05 mg/ml -45 C -70 D -55 -65 -90 190 200 210 220 230 240 250 260 190 200 210 Wavelength (nm) 220 230 240 250 Wavelength (nm) Figure 7. CD spectra of the peptides at various concentrations in 10 mM CaCl2 solution. (A) P1; (B) P2;(C)P3; (D) P4. The concentration dependent CD spectra of the peptides at 25 °C in CaCl2 solution are given in Figure 7. The CD spectra showed that the peptides (P1 to P3) gave a negative minimum at around 198-200 nm [9] at low concentrations, indicating that they have a random coil conformation. As the concentration increases, the negative minimum was red shifted (2 to 9 nm) and a observed shoulder between 225-228 nm indicate the presence of β-turn conformation. It was reported that the incorporation of proline would lead to an - 43 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 260 introduction in the turn-forming motif [12], which supported the above results. The intensity of the negative bands for all the peptides at the same concentration were about the same, which indicates that the peptides gave a more ordered structures in CaCl2 solution than in water (Figure 6). P4 gave different CD spectra as compared to the other peptides (P1 to P3); β-sheet conformation was obtained at both low and high concentrations of the peptide as reported by Zhang et al [13]. Similar to the other peptides, the intensity for P4 in CaCl2 solution is higher than in water. This shows that the divalent ion has an influence in the self-assembly for all the peptides providing a more ordered structure as compared to the free peptide. Quantitative analysis of the peptides at 2 mg/mL in 10 mM CaCl2 solution using the CD Spectra Devolution Software (CDNN) was summarized in Table 3. % Conformations for the peptides Peptides Conformation α-helix β-sheet β-turn Random coil P1 P2 P3 P4 5.8 34.1 20.9 36.7 9.1 41.9 19.9 32.7 9.0 41.9 19.8 33.1 7.3 46.2 17.0 32.1 Table 3. Quantitative analysis of the peptides at 2 mg/mL in 10 mM CaCl2 solution using CDNN software. - 44 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 3.3.2.3 Effect of salt solutions on solution conformations of the peptides 30 20 10 0 -10 -20 -50 CD (mdeg) CD (mdeg) -30 P1 (CaCl2 ) -70 -40 P2 (CaCl2) -60 -90 -80 P1 (NaCl) -110 A -130 -150 P2 (NaCl) B -100 -120 190 200 210 220 230 240 Wavelength (nm) 250 260 190 200 220 230 240 250 260 Wavelength (nm) 15 40 10 20 P3 (CaCl2) 5 0 P4 (NaCl) 0 -20 CD (mdeg) CD (mdeg) 210 -40 -60 -5 -10 -15 -80 P3 (NaCl) -20 C -100 -120 190 200 210 220 230 240 250 D P4 (CaCl2 ) -25 260 -30 190 200 210 Wavelength (nm) 220 230 240 250 Wavelength (nm) Figure 8. CD spectra of the peptides at 1 mg/mL in 10 mM of NaCl and CaCl2 solution respectively. (A) P1; (B) P2; (C) P3; (D) P4. Previous studies with alternating amphiphilic-peptide polymers and oligopeptides [1419] have shown that these polymers can adopt β-sheet structures and can aggregate, depending upon pH, salt and time allowed for the experiment. But as shown in Figure 8, only P4 has the ability to form β-sheet structure in both salt solutions [13], even though P1 and P2 were also arranged in alternating hydrophilic and hydrophobic amino acid - 45 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 260 residues. The other peptides gave a random coil conformation with a negative minimum between 204 and 205 nm [9] and positive maxima between 191-201 nm. Presence of a shoulder at 225 nm indicates a bend or β-turn conformation [12]. Peptides P1 to P3 form more ordered structures in NaCl solution than in CaCl2 solution, which indicates that Na+ exert a greater influence on the carboxylate groups of the acidic residues as compared to the divalent ions. This could be due to the smaller ionic radius of the monovalent ions, resulting in a higher ionic strength in the interaction between Na+ and the carboxylate ions as ionic strength can inhibit the structural transition [20]. Table 4 represent the quantitative analysis of the peptides at 1 mg/mL in 10 mM NaCl and CaCl2 solutions using the CD Spectra Devolution Software (CDNN). % Conformations for the peptides Peptides Conformation α-helix β-sheet β-turn Random coil P1 NaCl 12.7 30.5 23.2 33.6 P1 CaCl2 7.4 37.7 22.5 35.7 P2 NaCl 12.3 32.6 23.0 32.6 P2 CaCl2 7.6 37.9 22.7 35.0 P3 NaCl 15.2 29.6 23.5 31.8 P3 CaCl2 5.9 39.4 21.0 34.2 P4 NaCl 8.4 45.2 18.3 33.0 P4 CaCl2 6.7 47.4 17.4 31.2 Table 4. Quantitative analysis of the peptides at 1 mg/mL in 10 mM NaCl and CaCl2 solution using CDNN software. - 46 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 3.3.2.4 pH dependent 0 -6 -10 -30 CD (mdeg) CD (mdeg) -16 P1 (pH 5) -20 P1 (pH 3) -40 P2 (pH 5) -26 P2 (pH 3) -36 -50 -46 -60 A -70 B -56 -66 -80 190 200 210 220 230 240 250 190 260 210 230 250 Wavelength (nm) Wavelength (nm) 20 30 10 0 P4 (pH 3) 20 CD (mdeg) CD (mdeg) -10 -20 -30 -40 -50 10 0 P4 (pH 5) P3 (pH 5) -60 -10 C P3 (pH 3) -70 D -20 -80 190 200 210 220 230 Wavelength (nm) 240 250 260 190 210 230 250 Wavelength (nm) Figure 9. CD spectra of the peptides at 1 mg/mL in water at pH ∼ 3 and 5 at 25 °C. (A) P1; (B) P2; (C) P3; (D) P4. The pH effects for all the peptides were also investigated as shown in Figure 9. The two pH values showed a significant change in the particle size distributions for all the peptides, hence it was used to investigate the secondary structure conformations. Several studies of ionic self-complementary oligopeptides at various pH values showed the - 47 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com formation of β-sheet conformation that was quite stable over a wide range of pH [21]. But P1 and P2 did not give a β-sheet conformation even though they are incorporated with alternating hydrophobic and hydrophilic amino acid residues. Both of them gave a random coil conformation with a minimum between 195-198 nm [9]. This implies that P1 and P2 were not significantly affected by pH changes. P4 was able to give β-sheet conformation (negative minimum at 216 nm and positive maximum at 196 nm) due to the arrangement of the amino acid residues. P4 was arranged in alternating hydrophobic and hydrophilic manner but it has alternating positive and negative charges, which was not observed for the other two peptides (P1 and P2). The slight shift of the minimal ellipiticity is probably due to a change from ionic interaction between the side-chain to hydrogen bonding due to the neutralization of some of the ionic species. This may result in the slight changes in structure (Figure 9D). The intensities of the CD negative bands for P1 to P3 do not show any significant differences, which indicate that these peptides have a stable structure regardless of pH. The quantitative analysis of the peptides at 1 mg/mL in water at pH ∼ 3 and 5 using the CD Spectra Devolution Software (CDNN) was shown in Table 5. % Conformations for the peptides Peptides Conformation α-helix β-sheet β-turn Random coil P1 pH∼ 3 5.8 28.0 24.0 39.8 P2 pH∼ 3 6.1 40.4 20.1 33.7 P3 pH∼ 3 5.1 30.4 23.3 39.3 P4 pH∼ 3 4.8 46.4 18.4 34.9 P1 pH∼ 5 5.4 30.8 22.3 38.5 P2 pH∼ 5 6.0 39.4 20.5 33.9 P3 pH∼ 5 5.0 26.2 24.2 41.5 P4 pH∼ 5 4.4 47.6 18.4 35.0 Table 5. Quantitative analysis of the peptides at 1 mg/mL in water at pH ∼ 3 and 5 at using CDNN software. - 48 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 3.4 Atomic force microscopy studies of the peptides (P1 – P4) Atomic Force Microscopy (AFM) was used to gain information about the morphology and the self-assembly of the peptides under different conditions on the hydrophilic mica surface. As observed from the DLS data, there is a great change in the particle size distribution at pH ∼ 3 and 5. Therefore, these two pH values were chosen to test the effect on the self-assembled nanostructures, and two types of morphologies were observed. 3.4.1 Self-assembly of peptides in water at high concentration (1 mg/mL) at pH ∼ 3 and 5 Peptide 1: ADAELDPRGDFPDLEADA A - 49 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com B Figure 10. Self-assembly of P1 adsorbed onto mica substrate from water. Images are AFM scans (the brightness of features increases as a function of height) of a freshly cleaved mica surface over which it was immersed in the P1 solution for about 15 mins. (A) at pH ∼ 3; (B) at pH ∼ 5. At pH ∼ 3, globular assemblies with a diameter of about 25-30 nm is observed. Upon increasing the pH to 5, no globular assemblies were detected but worm-like structure with a strand diameter of about 15-20 nm was observed. From studies using CD spectroscopy, both pH values gave a random coil with a bend or β-turn conformation. - 50 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Peptide 2: IELKPRGDFPDLEA A B Figure 11. Self-assembly of P2 onto mica substrate from water. Images are AFM scans (the brightness of features increases as a function of height) of a freshly cleaved mica surface over which it was immersed in the P2 solution for about 15 mins. (A) at pH ∼ 3; (B) at pH ∼ 5. AFM images for P2 are given in Figure 11. The calculated pI for this peptide is 4.32. Regardless of the pH, this peptide gave spherical assemblies on the mica surface. The - 51 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com AFM images show that the spherical islands formed are larger than P1 at pH ∼ 3 with a particle size of ∼ 35 nm. As the pH is increased to 5, no worm-like structures were observed but small spherical particles with diameter of about 25-30 nm were seen. As in the same case as P1, there is no significant change in the secondary structures investigated by DLS and CD spectroscopy at the two pH values. Both P1 and P2 are incorporated with hydrophilic and hydrophobic amino acid residues and RGD in the middle of the sequence. The observed morphologies of these two peptides are somewhat different at pH ∼ 5. But at pH ∼ 3, the morphology of P1 and P2 were about the same except the particle sizes are different. - 52 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Peptide 3: LPLLKGDLRI A B Figure 12. Self-assembly of P3 onto mica substrate from water. Images are AFM scans (the brightness of features increases as a function of height) of a freshly cleaved mica surface over which it was immersed in the P3 solution for about 15 mins. (A) at pH ∼ 3; (B) at pH ∼ 5. Peptide P3 gave a pI of 8.75 and the above figure (Figure 12) represents the selfassembly of P3 on the mica surface at two different pH values. The appearance of - 53 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com identical morphology at two different pH indicates that self- assembly of P3 is independent of pH as can be seen in the CD spectra (Figure 9C). The morphology obtained on the mica substrate show a worm-like structure. The diameter of the wormlike structure at pH ∼ 3 and pH ∼ 5 were about the same (∼ 29 nm). Since the two pH values are above the isoelectric point, we obtained similar morphologies as the overall charges for this peptide at these two pH values are positive which can interact with the negatively charged mica substrate. As observed in the CD spectrum, the secondary structures for P3 at both pH values are the same. As mentioned previously, the self-assembly of peptide P4 has been reported by Zhang et al. [13, 20-21]. It consists of alternating hydrophobic and hydrophilic amino acids; they are highly soluble in pure water and have the tendency to form an unusually stable βsheet structure. From the CD spectra (Figure 9D), P4 forms a β-sheet structure and are capable of forming fiber-like structures. Figure 13 shows the AFM images of P4 in water at two different pH. In the self-assembly of P4, fiber-like structures are obtained at both pH values on the hydrophilic mica. The diameter of the fibers for pH ∼ 3 is ∼ 30 nm and for pH ∼ 5 is ∼ 15 nm. In general, short oligopeptides, such as 16 mer, are not considered as building blocks for biomaterials, and most oligopeptides do not form regular or stable structures but as seen from our results, we do obtain rather stable structures. - 54 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Peptide 4: RADARADARADARADA A B Figure 13. Self-assembly of P4 onto mica substrate from water. Images are AFM scans (the brightness of features increases as a function of height) of a freshly cleaved mica surface over which it was immersed in the P4 solution for about 15 mins. (A) at pH ∼ 3; (B) at pH ∼ 5. - 55 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com In conclusions, P1 at pH ∼ 3 and P2 at both pH values have the same kind of morphologies. The only difference is when the pH is changed to 5 for P1, where wormlike structure was observed, which is consistent with the DLS and CD data. The particle size distribution for P1 at pH ∼ 5 was much larger compared to P2 and the intensity from the CD data (Figure 9A) at this particular pH for P1 was also much higher indicating that more ordered structure was formed. As compared to P1 and P2, P3 and P4 were found to have the same morphologies. This is interesting because P4 was arranged in similar manner as P1 and P2, but P4 forms fiber-like structure on mica substrate. This can be due to the stable β-sheet structure that was formed by P4, which was capable of forming fiber-like morphology. - 56 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 3.4.2 Influence of salt solutions on the self-assembly of peptides The effect of self-assembly of the peptides in salt solutions was investigated, and two types of morphologies were observed - spherical particles and fiber-like structures. The AFM images showed that monovalent ions (Na+) have a weaker influence than Ca2+ because Ca2+ needs to bind to two carboxylate groups and hence resulting in a compact structure. Peptide 1 A - 57 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com B Figure 14. Self-assembly of P1 on mica in presence of metal ions. Images are AFM scans (the brightness of features increases as a function of height) of a freshly cleaved mica surface over which the peptide P1 was self-assemble from (A) CaCl2 solution; (B) NaCl solution on mica for about 30 mins. Peptide 2 A - 58 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com B Figure 15. Self-assembly of P2 on mica in presence of metal ions. Images are AFM scans (the brightness of features increases as a function of height) of a freshly cleaved mica surface over which the peptide P2 was self-assemble from (A) CaCl2 solution; (B) NaCl solution on mica for about 30 mins. The effects of the self-assembly of the peptides on mica in the presence of metal ions were also studied. In the presence of Na+ ions (Figure 14B), a fiber-like structure was observed and small islands of diameter 20-30 nm were formed in presence of Ca2+ (Figure 14A) on mica substrate for P1. For P2, spherical aggregates are observed with irregular sizes in NaCl solution. The same morphology was obtained in CaCl2 solution for P2 (Figure 15A) but the size was twice that of P1 (∼ 44 nm). Overall, there is no significant difference in morphology for P1 and P2 in presence of Ca2+ ions, which is consistent with the results obtained from DLS and CD spectra. - 59 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Peptide 3 A B Figure 16. Self-assembly of P3 on mica in presence of metal ions. Images are AFM scans (the brightness of features increases as a function of height) of a freshly cleaved mica surface over which the peptide P3 was self-assemble from (A) CaCl2 solution; (B) NaCl solution on mica for about 30 mins. The above figure (Figure 16) represents the self-assembly of P3 on mica in the presence of Na+ or Ca2+ ions. In addition to the spherical aggregates, fiber-like structures were also - 60 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com observed in the presence of Ca2+ ions (Figure 16A). In the presence of Na+, P3 assembles uniformly, which agrees well with the DLS results where highly monodisperse particles are formed. The diameter for the worm-like structures was about 58.8 nm and the spherical islands were about 44.1 nm in CaCl2 solution. Peptide 4 A B Figure 17. Self-assembly of P4 on mica in presence of metal ions. Images are AFM scans (the brightness of features increases as a function of height) of a freshly cleaved - 61 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com mica surface over which it was immersed in the P4 solution for about 30 mins. (A) in CaCl2 solution; (B) in NaCl solution. Both images showed fiber-like morphologies, which were the same as the AFM images obtained at two pH values for the same peptide. P4 in presence of Na+ (Figure 17B) gave a densely covered film as the duration of the immersion time (∼ 30 mins) of the mica in the peptide solution might be too long. The measured diameter of the fiber in the presence of Ca2+ ions (Figure 17A) is about 73.5 nm and that in NaCl solution is about 58.8 nm. In conclusions, there is no significant difference in morphology for P1 and P2 in the presence of Ca2+ ions. Both have the same intensity in CaCl2 solution, resulting in similar morphology. The intensity of P1 in NaCl is higher than P2 in the CD spectra, resulting in a more ordered structure formed for P1 even though the particle size distribution for P1 and P2 are about the same. Fiber-like structures were formed by P4. Even though peptides P1, P2 and P4 consist of alternating hydrophobic and hydrophilic amino acid residues, only P4 forms fiber-like structure in both salt solutions. This could be due to the presence of the proline in P1 and P2, which limits the conformational flexibility of the peptide chain. Even though, P3 has the same conformation as P1 and P2, it has a different type of morphology as compared to these peptides. One reason might be due to the many hydrophobic amino acid residues present in P3, which may favor pH or metal ion assisted aggregation. - 62 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com It is possible to have smaller structures in solutions and slightly bigger architecture in the solid state due to the different mechanism of formation and stabilization. In solutions, the structures were stabilized through solvation. Also, the nucleation and growth process was slow. However on the solid substrate, due to the fast evaporation of the solvent, the nucleation and growth of the structures was relatively fast. The aggregation size also increases due to the large concentration changes from the rapid solvent evaporation. So the data collected from both the DLS and AFM structures are consistent. Despite the widespread use of mica, the mechanism of adsorption of peptides is not well understood. For both glass and mica in aqueous media, it is known that positive ions tend to dissociate from the surface to make substrate negatively charged at neutral pH and that the neutrality can be altered by changing the pH of the buffer solution [22]. It is plausible to assume that electrostatic interaction is primarily responsible for adsorption. However, we must also realize that most of these charged groups are shielded by counter-ions in solution. Therefore, it is not clear whether it is the direct interaction between the oppositely charged groups or the salt bridges between like charged groups that is responsible for surface adsorption in each case. The figure below shows the two possible mechanisms for surface adsorption of macromolecules in aqueous media. - - - - + + + ++++ - - - - - +++++ - + - - - - ++ ++ ++ - - - Figure 18. Two possible mechanisms for surface adsorption of macromolecules in aqueous media. On the left, a direct charge interaction is shown to tether the molecule to the negatively charged substrate surface. On the right, an intermediate divalent cation is - 63 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com shown to mediate the adsorption of a negatively charged molecule to a negatively charged substrate. These are probably the two major means for adsorbing biological macromolecules to a hydrophilic substrate, such as mica [22]. 3.5 Effects of the calcite crystals morphologies in the presence of peptides 3.5.1 Scanning Electron Microscopy (SEM) Mineralization in living organisms generates structures different from abiotic crystal growth. Mimicking biomineralization in in vitro may be a way of synthesizing inorganic materials with useful microstructures. Most of the earlier works using synthetic templates have addressed the role of acidic macromolecules or short peptides with acidic and neutral repeating residues on the CaCO3 crystallization [23-24]. Their observation revealed that the polymorph specificity depends on the amino acid sequence, the conformation of specific peptides or proteins and the microenvironment of crystal nucleation and growth [25-26]. Many biomineral-related proteins can be considered to possess the characteristic intermolecular association properties. These will include phosphophoryn [27], and amelogenin [28], which are involved in apatite mineralization, silaffins in biosilica morphogenesis [29] and ansocalcin in eggshell calcification [30]. Our group has successfully synthesized a few designer oligopeptides, which induce the nucleation of a few interesting calcite crystals morphologies [31]. These peptides were incorporated with charge amino acid residues in multiplets, which gave a stable and well-defined secondary structure and consisted of many charged amino acid multiplets. During the course of this project, a few peptides were designed to study the - 64 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com formation of secondary structures such as α-helix and β-sheet that can act as an ideal template for calcite nucleation [32]. Peptide 1: ADAELDPRGDFPDLEADA A B C D E F Figure 19. SEM micrographs of calcite crystals in the presence of P1 at various concentrations. (A) Control; (B) 2 mg/mL; (C) 1 mg/mL; (D) 500 µg/mL; (E) 100 µg/mL; (F) 50 µg/mL. - 65 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Figure 19 shows the scanning electron micrographs revealing the changes in the morphology of calcite crystals grown in the presence of P1. The CaCO3 crystals were grown on microscopy slides by slow diffusion of CO2 (generated by the decomposition of ammonium carbonate) to the peptide dissolved in 7.5 mM CaCl2 solution and left it for 2 days inside a sealed desiccator. In the control experiments with no added peptide, exclusive nucleation of rhombohedral calcite crystals was observed (Figure 19A). At low concentrations (Figure 19E–F) of the peptide P1, the sharp rhombohedral edges of the calcite crystals remained, which was the same as the control. This might be due to the low concentration of the peptide present in the solution which is not strong enough to induce any change in the calcite crystals. But as the concentration increases (Figure 19BD), “hopper” crystals was formed due to the non-specific binding of this peptide with the calcium carbonate crystals [33]. No significant aggregation of the calcite crystals was observed at high concentration (2 mg/ml) which implied that this peptide was not active enough to induce calcite nucleation. The scanning electron micrographs of the calcite crystals grown in the presence of P2 were shown in Figure 20. Without the presence of P2, well-defined rhombohedral calcite crystals were observed. Similar to P1, P2 also did not show any major effect on the calcite crystal morphologies. As a whole, these two peptides (P1 and P2) do not have any significant effect on the morphology of the calcite crystals as no aggregation or any polymorph selectivity was observed, even though both the peptides contain aspartic acid, which can interact with the calcium ions [34]. - 66 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Peptide 2: IELKPRGDFPDLEA A B C D E F Figure 20. SEM micrographs of calcite crystals in the presence of P2 at various concentrations. (A) Control; (B) 2 mg/mL; (C) 1 mg/mL; (D) 500 µg/mL; (E) 100 µg/mL; (F) 50 µg/mL. - 67 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Peptide 3: LPLLKGDLRI A A B F E C E D F Figure 21. SEM micrographs of calcite crystals in the presence of P3 at various concentrations. (A) Control; (B) 2 mg/mL; (C) 1 mg/mL; (D) 500 µg/mL; (E) 100 µg/mL; (F) 50 µg/mL. Scanning electron micrographs of the changes in the morphology of the calcite crystals in the presence of P3 are given in Figure 21. As compared to the control, some steps are formed at 1 mg/mL (Figure 21C). As the concentration decreases (Figure 21D-E), the - 68 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com calcite crystals start to aggregate, but it is not significant as the other peptides [31]. At the lowest concentration (Figure 21F), the morphology of the calcite crystals is same as in Figure 21A (control), which does not have any added peptide. This indicate that P3 does play a role in the nucleation of CaCO3 crystals but not as strong as the other peptides [31]. The scanning electron micrographs (Figure 22) showed the changes in the morphology of calcite crystals grown in the presence of P4. As with all the other peptides, P4 is not an exception, P4 does not induce significant changes in calcite crystal morphology or aggregate formation in in vitro crystallization experiments. As the concentrations decreases, there is no significant change in morphology, even though P4 was found to have a stable secondary structure as shown in Figure 7D. This may be because the amino acid residues for P4 were arranged in an alternating hydrophobic and hydrophilic manner [16], and there were no repeating acidic multiplets necessary for inducing calcite aggregation and nucleation. - 69 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Peptide 4: RADARADARADARADA A A B C D E F Figure 22. SEM micrographs of calcite crystals in the presence of P4 at various concentrations. (A) Control; (B) 2 mg/mL; (C) 1 mg/mL; (D) 500 µg/mL; (E) 100 µg/mL; (F) 50 µg/mL. In conclusions, no aggregations are observed in the morphologies of the calcite crystals in the presence of these four peptides; only “hopper” crystals in the presence of P1 and P2 and weak aggregation of crystals in the presence of P3 were observed. This may be due - 70 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com to the arrangement of the amino acid residues (alternating hydrophobic and hydrophilic amino acid residues) on the peptide backbone. These results strongly support the importance of charged residue multiplets in calcification of the goose eggshell reported earlier by our group [30]. It was found that multiplets of acidic and basic amino acid residues were needed to induce calcite crystal aggregates as seen from the sequence of ansocalcin protein obtained from the goose eggshell and in the designer peptides (REWD16 and REWDP17) [31]. This protein and the two designer peptides showed extensive aggregation as the concentration increases. The peptides P1 to P4 lack such multiplets of charged amino acid residues and were therefore inactive towards CaCO3 crystallization. - 71 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 3.5.2 Energy Dispersive X-ray Scattering (EDXS) To determine the types of elements present on the crystal surfaces, energy dispersive Xray scattering was carried out. A B C D Figure 23. EDXS spectra of the crystal surface of the four peptides (P1 to P4) at 2 mg/mL: (A) P1; (B) P2; (C) P3; (D) P4. Figure 23 represents the energy dispersive X-ray scattering spectrum for all the four peptides. The atomic percentage of calcium in the single crystal was about 12 % for P1 (Figure 23A). As in the cases of the other peptides, the amount of calcium on the crystal surface was found to be about 16 % for P2 (Figure 23B); 15 % for P3 and 13 % for P4 - 72 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com (Figure 23D). The presence of the Pt peak was resulted due to the coating of the calcite crystals when taken for SEM imaging. From these EDXS spectra, we can deduce that calcium is present on the surface of the crystals for all the four peptides. 3.5.3 Powder X-Ray Diffraction (XRD) {104} Peptide 1 Intensity (a.u.) Peptide 2 Peptide 3 Peptide 4 20 30 40 50 60 70 Wavelength (nm) Figure 24. X-ray diffraction of single crystals formed at 2 mg/mL of the four peptides (P1 to P4). The XRD spectra of the CaCO3 crystals grown in the presence of the four peptides at a concentration of 2 mg/mL are shown in Figure 24 indicating the presence of calcite phase. All these peptides have a peak with high intensity at 2θ = 29 degrees, which corresponds to the {104} face indicating that the preferred orientation of the crystals formed. Diffractions from all other crystal planes were also observed indicating single crystalline nature of the crystal aggregates. But no aggregations were observed in all the - 73 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com peptides synthesized indicating that it could be due to the arrangement of the amino acid residues and the lack of charged residue multiplets. 3.6 References 1. Brahmachari, S. K.; Rapaka, R. S.; Bhatnagar, R. S.; Ananthanarayanan, V. S. Biopolymers 1982, 21, 1107-1125. 2. Hollosi, M.; Kawai, M.; Fasman, G. D. Biopolymers 1985, 24, 211-242. 3. Rose, G. D.; Gierasch, L. M.; Smith, J. A. Adv. Protein Chem. 1985, 37, 1-109. 4. Gorman, J. Science News 2000, 158, 364-367. 5. (a) MacArthur, M. W.; Thornton, J. M. J. Mol. Biol. 1991, 218, 397-412 (b) Harkey, M. A.; Klueg, K.; Shepperd, P.; Raff, R. A. DeV. Biol. 1995, 168, 549566 (c) Benson, S. C.; Wilt, F. H. In Calcification in Biological Systems; Bonucci E., Ed.; CRC Press: Boca Raton, FL, 1992, 157-178. 6. (a) Moradian-Oldak, J.; Leung, W.; Fincham, A. G. Biopolymers 1998, 46, 225238 (b) Moradian-Oldak, J.; Leung, W.; Fincham, A. G. J. Struct. Biol. 1998, 122, 320-327 (c) Killian, C. E.; Wilt, F. H. J. Biol. Chem. 1996, 271, 9150-9159 (d) Wustman, B. A.; Morse, D. E.; Evans, J. S. Langmuir 2002, 18, 9901-9906 (e) Wustman, B. A.; Santos, R.; Zhang, B.; Evans, J. S. Biopolymers 2002, 65, 362372 (f) Zhang, B.; Wustman, B. A.; Morse, D. E.; Evans, J. S. Biopolymers 2002, 63, 358-369 (g) Zhang, B.; Xu, G. Z.; Evans, J. S. Biopolymers 2000, 54, 464-475 (h) Xu, G. Z.; Evans, J. S. Biopolymers 1999, 49, 303-312. 7. Bergdoll, M.; Remy, M. H.; Cagnon, C.; Masson, J. M., Dumas, P. Structure 1997, 5, 391-398. - 74 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 8. Ruoslahti, E.; Pierschbacher, M. D. Science 1987, 238, 491-497. 9. Zhang, B.; Wustman, B. A.; Morse, D.; Evans, J.S. Bioploymers 2002, 63, 358369. 10. Marqusee, S.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8898-8902. 11. Marqusee, S.; Robbins, V. H.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5286-5290. 12. Jayakumar, R.; Jayanthy, C.; Gomathy, L. Int. J. Peptide Protein Res. 45, 1995, 129-137. 13. Zhang, S.; Altman, M. React. Funct. Polym. 1999, 41, 91-102. 14. Brack,A.; Orgel, L. E. Nature (London) 1975, 256, 383-387. 15. Rippon, W. B.; Chen, H. H.; Wagner, A. G. J. Mol. Biol. 1973, 75, 369-375. 16. Seipke, G.; Arfmann, H. A.; Wagner, K. G. Biopolymers 1974, 13, 1621-1633. 17. Piggion, E.; Cosani, A.; Terbojevich, M.; Borin, G. Biopolymers 1972, 11, 633643. 18. St. Pierre, S; Ingwall, R. T.; Varlander, M. S.; Goodman, M. Biopolymers 1978, 17, 1837-1847. 19. Osterman, D. G.; Kaiser, E. T. J. Cell. Biochem. 1985, 29, 57-72. 20. Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3334-3338. 21. Zhang, S.; Rich, A. Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 23-28. 22. Melander, W.; Horvath, C. Arch. Biochem. Biophys. 1977, 183, 200-215. 23. (a) Wheeler, A. P.; Sikes, C. S. In Chemical Aspects of Regulation of Mineralization; Sikes, C. S., Wheeler, A. P., Eds; Mobile, AL, 1988, 9-13 (b) - 75 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Wheeler, A. P.; Sikes, C. S. In Material Synthesis Utilising Biological Process; Rieke, P. C., Calvert, P. D., Alper, M., Eds; Material Research Society: Pittsburgh, PA, 1989, 45-50. 24. Wierzbicki, A.; Sikes, C. S.; Madura, J. D.; Drake, B. Calcif. Tissue Int. 1994, 54, 133-141. 25. Levi, Y.; Albeck, S.; Brack, A.; Weiner, S.; Addadi, L. Chem. Eur. J. 1998, 4, 389-396. 26. DeOliveira, D. B.; Laursen, R. A. J. Am. Chem. Soc. 1997, 119, 10627-10631. 27. Stetler-Stevenson, W. G.; Veis, A. Calc.Tissue Int. 1987, 40, 97-102. 28. Moradian-Oldak, J. Matrix Biol. 2001, 20, 293-305. 29. Kroger, N.; Lorenz, S.; Brunner, E.; Sumper, M. Science 2002, 298, 584-586. 30. Lakshminarayanan, R.; Kini, R. M.; Valiyaveettil, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5155-5159. 31. Ajikumar, P. K.; Lakshminarayanan, R.; Ong, B. T.; Valiyaveettil, S.; Kini, R. M. Biomacromolecules 2003, In press. 32. Addai, L.; Weiner, S. Angew. Chem., Int. Ed. Engl. 1992, 131, 153-169. 33. Gower, L. A.; Tirrell, D. A. J. Crystal Growth 1998, 191, 153-160. 34. Kato, T.; Sugawara, A.; Hosoda, N. Adv. Mater. 2002, 14, 869-877. 35. Gregoire, C.; Marco, S.; Thimonier, J.; Duplan, L.; Laurine, E.; Chauvin, J. P.; Michel, B.; Peyrot, V.; Verdier, J. M. The EMBO J. 2001, 20, 3313-3321. - 76 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com CHAPTER 4 CONCLUSIONS - 77 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com CHAPTER 4: CONCLUSIONS 4.1 Conclusions Four peptides were designed with specific sequences to understand the self-assembly and biomineralization processes. The DLS results showed that the particle size distributions for all the peptides increased as the pH increase from 3 to 5. The same trend was observed in the salt solutions; the peptides in NaCl solution have a larger particle size distribution than in CaCl2 solution. In the CD spectra, all the peptides except P4 gave a random coil with the presence of a bend/β-turn conformation, whereas P4 gave a β-sheet structure in both salt solutions and at different pH. This finding of P4 agree well with the previous studies that peptides consisting of alternating hydrophilic and hydrophobic amino acid residues have a tendency to adopt a β-sheet structure, which states that they are able to form stable β-sheets in the presence of salt, various pHs and prolonged incubation [1-8]. Surprisingly, P1 and P2 did not exhibit β-sheet structures even though both of them are arranged in alternating hydrophobic and hydrophilic fashion. Hence apart from arranging them in alternating hydrophobic and hydrophilic layout, additional information such as the type of amino acids to use, the degree of intermolecular interaction and the peptide length will have an impact on the secondary structures obtained. AFM studies were done under different pH (3 and 5) in water and salt solutions to observe any changes in the self-assembly for all the peptides. Interesting morphologies were obtained for both pHs and in CaCl2 solution, which can be explained in terms of - 78 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com their secondary structures. They can undergo changes in their secondary structures in different solvent environment, such as varying pH or salt concentration. It was found that peptide 3 (P3) and peptide 4 (P4) gave fiber-like structures, whereas spherical particles were observed for the peptides (P1 and P2). The key for success in AFM is to use very low concentrations of protein (preferably in the range of micrograms per milliliter) in a buffer to prevent aggregation of the peptide. In most cases, milligrams per milliliter or higher concentrations were used, which resulted in a large number of loosely attached molecules on the surface. When the atomic force microscope tip was engaged, it was readily contaminated, showing no contrast in the image. In this case, one normally assumes no adsorption and would use even more proteins [9]. Low concentration and a controlled incubation time appeared to be a better approach in this work. In the second part of this work, no interesting morphologies were observed on the calcium carbonate crystals. This showed that the designed peptides do not induce any polymorph selectivity on the biomineralization process. P1, P2 and P4 all contained hydrophilic and hydrophobic residues while P3 contained mostly hydrophobic residues. It has been shown that soluble proteins involved in biomineralization contain large amounts of aspartic acid [10-12]. Therefore, it is expected that the amino acids that have carboxyl groups can interact with the calcium ions, and will exert an effect on the calcite crystallization. In order to better understand the mechanism behind calcite crystallization, a few acidic amino acid residues were incorporated on the peptides. Earlier investigations - 79 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com from our laboratory have shown that large amounts of acidic groups on the template are involved in biomineralization. But in our findings, even in the presence of acidic amino acid residues, there was no significant effect on the calcium carbonate morphologies. This may lead us to conclude that multiplets of acidic amino acid residues and the organization of them along the peptide sequence may be important to induce significant change in the morphologies [13-14]. Previous studies have suggested that peptides with a secondary structure that preferentially interacts with calcium carbonate can have an effect in the calcite crystallization morphology [15-17]. These peptides have either an α-helix or β-sheet conformations. As expected, no aggregation or nucleation of calcite crystals were observed for peptides P1 to P3, since they do not have a stable and well-defined conformation. But from the CD results, P4 have a stable β-sheet conformation and it also contained acidic amino acid residues, but it does not induce any interesting morphologies. Kato et al. [18] said that the position and distance of the carboxylic acids in macromolecules were important as they may cooperate to bind calcium ions. Hence this might be the reason why P4 does not induce any calcite morphologies. 4.2 References 1. Brack, A; Orgel, L. E. Nature 1975, 256, 383-387. 2. Brack, A; Caille, A. Int. J. Protein Res. 1978, 11, 128-139. 3. Seipke, G; Arfmann, H. A.; Wagner, K. G. Biopolymers 1974, 13, 1621-1633. 4. Piggion, E; Cosani, A; Terbojevich, M; Borin, G. Biopolymers 1972, 11, 633-643. - 80 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 5. Rippon, W. B.; Chen, H. H.; Walton, A. G. J. Mol. Biol. 1973, 75, 369-375. 6. St. Pierre, S; Ingwall, R. T.; Varlander, M. S.; Goodman, M. Biopolymers 1978, 17, 1837-1847. 7. Trudelle, Y. Polymer 1975, 16, 9-15. 8. Osterman, D. G.; Kasier, E. T. J. Cell. Biochem. 1985, 29, 57-72. 9. Heuser, J. J. Electron Microsc. Techniques 1989, 13, 244-263. 10. Addadi, L; Weiner, S. Angew. Chem. Int. Ed. Engl. 1992, 31, 153-169. 11. Mann, S. Nature 1988, 332, 119-124. 12. Weiner, S; Addadi, L. J. Mater. Chem. 1997, 7, 689-702. 13. Lakshminarayanan, R.; Kini, R. M.; Valiyaveettil, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5155-5159. 14. Ajikumar, P. K.; Lakshminarayanan, R.; Ong, B. T.; Valiyaveettil, S.; Kini, R. M. Biomacromolecules 2003, In press. 15. Wen, D; Laursen, R. A. Biophys. J. 1992, 63, 1659-1662. 16. Wen, D; Laursen, R. A. J. Biol. Chem. 1992, 267, 14102-14108. 17. Donners, Jack J. J. M.; Nolte, Roeland J. M.; Sommerdijk, Nico A. J. M. J. Am. Chem. Soc. 2002, 124, 9700-9701. 18. Kato, T; Sugawara, A; Hosoda, N. Adv. Mater. 2002, 14, 869-877. - 81 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com [...]... this direction we designed and investigated the self-assembly of a few novel peptides at different conditions Herein we report the design strategy, synthesis and characterization of four peptides and their self-assemblies in different environments and the role in the crystallization of CaCO3 Two areas were studied in this research: (1) self-assembly of the peptides and (2) understanding of the protein-mineral... [63] Three of the peptides synthesized consist of alternating hydrophobic and hydrophilic residues Out of these three peptides, two of them consist of a cell adhesion motif ‘RGD’ in the middle of the peptide This tripeptide motif is a well studied and an important ligand for some members of the integrin family of the cell adhesion receptors The fourth peptide is incorporated with a mimic of the cell... due to separation and purification is avoided The insoluble polymer can be filtered and washed without losses [1] Solid-phase peptide synthesis consists of three distinct sets of operations: (1) chain assembly of peptide chain on a resin; (2) simultaneous or sequential cleavage and deprotection of the resin-bound, fully protected peptide chain; and (3) purification and characterization of the target peptide... images of P1 adsorbed onto mica substrate from water 49 at pH ~ 3 and 5 Figure 11 AFM images of P2 adsorbed onto mica substrate from water 51 at pH ~ 3 and 5 Figure 12 AFM images of P3 adsorbed onto mica substrate from water 53 at pH ~ 3 and 5 Figure 13 AFM images of P4 adsorbed onto mica substrate from water 55 at pH ~ 3 and 5 Figure 14 AFM images of P1 adsorbed onto mica substrate from 10 mM 57 CaCl2 and. .. are chemical and structural complementarity Like hands and gloves, both the size and the correct orientation, i.e chirality, are important in order to have a complementary and compatible organization Molecular self-assembly in nature Biomimicry and designing nature-inspired materials through molecular self-assembly is an emerging field of research in recent years Nature is a grand master at designing... inhibition of the process at other sites; 2) A specific mineral is produced with a defined crystal size and orientation; or 3) Macroscopic growth is accomplished by the incremental growth of unique biocomposites The effectiveness of the crystal growth and inhibition processes depends on the structure and chemistry of the interfaces between organic substrate, mineral, and medium The highly specific control of. .. are useful analogues of proteins and have been used extensively to probe the role of functional motifs in altering the kinetics of crystal growth processes [50-53] Based on the partial amino acid sequence available from the mollusk shells nacre, Levi et al synthesized a series of peptides containing hydrophobic and hydrophilic amino acids and found that the peptides with stretches of poly(Asp-Leu) domains... potential disadvantages of the stepwise SPPS involve incomplete reactions and the gradual buildup of insoluble by-products [60] - 11 PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com 1.4 Outline of Thesis 1.4.1 Aim and scope of present work Here in this present work, we designed and synthesized peptides which are expected to form stable secondary structures and interesting materials... spectra of the crystal surface of the four peptides (P1 to 72 P4) at 2 mg/mL Figure 24 XRD of single crystals formed at 2 mg/mL of the four peptides 73 (P1 to P4) - ix PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com Summary Molecular self-assembly is a unique and powerful method for assembling building blocks for functional materials and devices Self-assembly of nucleic acid and. .. formation of calcium salt crystals While beneficial for tooth or bone mineralization, precipitation of calcium salts can be extremely harmful in fluids because it leads to the formation of stones and to the development of a lithiasis A variety of minerals such as calcium carbonate, hydroxyapatite, silicate, and iron oxides are employed as biominerals The control of the crystal shape/morphology of calcium .. .DESIGN AND CHARACTERIZATION OF FUNCTIONAL NOVEL OLIGOPEPTIDES ONG BOON TEE (B.Sc (Hons.), NUS) A THESIS SUBMITTED FOE THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL... direction we designed and investigated the self-assembly of a few novel peptides at different conditions Herein we report the design strategy, synthesis and characterization of four peptides and their... Biomineralization 1.4 Outline of Thesis 1.5 1.4.1 Aim and scope of present work 12 References 13 Chapter 2: Synthesis and Characterization of Self-Assembly Peptides 18 2.1 19 Materials and Methods -iPDF