Structure to function: Spider silk and human collagen
vi TABLE OF CONTENTS ABSTRACT ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES
PART 1: SPIDER SILK BLOCK COPOLYMERS 1
CHAPTER 1: SPIDER SILKS
1.1 Spider Silks - Ideas for Nanoscience 3 1.1.1 Anatomy and Physiology of the Spider Spinning Apparatus (Nephila clavipes) 4 1.1.2 Spider Silk Types and Their Modules 7 184.108.40.206 Major Ampullate Silk Proteins 8 220.127.116.11 Minor Ampullate Silk Proteins 10 18.104.22.168 Flagelliform Silk Proteins 11 22.214.171.124 Aciniform Silk Proteins 12 126.96.36.199 Tubuliform (Eggcase) Silk Proteins 13 188.8.131.52 Fiber Coating (Glue) and Pyriform Silk Proteins 14 1.1.3 Secondary Structure Elements of Silk Proteins 16 1.1.4 Mechanical Properties of Spider Silks 23
CHAPTER 2: BLOCK COPOLYMERS
2.1 Principles of Self-assembly 25 2.1.1 The Role of Intermolecular Forces in Self-assembly 26 2.1.2 Thermodynamics of Self-assembly 27 2.2 Mechanisms of Block Copolymer Behavior 30 2.3 Hierarchical Self-assembly in Block Copolymers 35 2.4 Biocompatible Amphiphilic Block Copolymers 37
vii CHAPTER 3: SPIDER SILK BLOCK COPOLYMERS THROUGH THE RECOMBINANT DNA TECHNOLOGY
3.1 Design of Spider Silk Block Copolymers 45 3.2 Materials and Methods 47 3.2.1 Construction of cloning vector pET30L 44 3.2.2 Cloning of Silk Modules into a pET30L Vector 48 3.2.3 Expression of Spider Silk Block Copolymers 49 3.2.4 Purification of Spider Silk Block Copolymers 49 3.2.5 Purification Tag Removal 50 3.2.6 Protein Identification 51 3.3 Results and Discussion 51 3.3.1 Cloning of spider silk-like block copolymers 53 3.3.2 Expression and purification of spider silk-like block copolymers 56 3.4 Conclusion 60
CHAPTER 4: BIOPHYSICAL CHARACTERIZATION OF A NOVEL FAMILY OF SPIDER SILK BLOCK COPOLYMERS
4.1 Materials and Methods 62 4.1.1 Secondary Structure Analysis 62 4.1.2 Morphological Analysis 63 4.2 Results and Discussion 63 4.2.1 Secondary Structure Analysis 63 4.2.2 Morphology Analysis 70 4.3. Partial Phase Diagram of Spider Silk Block Copolymers 76 4.4 Potential Applications of Spider Silk Block Copolymers 79 4.5 Conclusion 81
CHAPTER 5: GENETIC ENGINEERING OF TAG-FREE SPIDER SILK CONSTRUCTS
5.1 Advantages of Tag-Free Genes 83 5.2 Materials and Methods 85
viii 5.2.1 Design and Cloning of Tag-Free Silk Genes 85 5.2.2 Expression and Purification of Tag-Free Silk Genes 86 5.3 Results and Discussion 88 5.3.1 Cloning of Tag-Free Silk Genes 88 5.3.2 Expression, Purification and Detection Challenges 90 5.4 Conclusion and Future Work 93
CHAPTER 6: COLLAGEN STRUCTURAL HIERARCHY AND SUSCEPTIBILITY TO DEGRADATION BY ULTRAVIOLET RADIATION
6.1 Structure and Function of Collagen Type I 96 6.2 Materials and Methods 98 6.2.1 Preparation of collagen films 98 6.2.2 Irradiation of collagen films 98 6.2.3 Material Characterization 99 6.3 Results and Discussion 100 6.3.1 Preparation of collagen solutions 100 6.3.2 FTIR spectroscopy 100 184.108.40.206 The behavior of the amide A band components 101 220.127.116.11 The behavior of Amide I band components 105 18.104.22.168 The behavior of Amide II band components 110 6.3.3 Polyacrylamide Gel Electrophoresis 112 6.3.4 Atomic Force Microscopy 113 6.3.4 Reactions Leading to Collagen Damage 116 22.214.171.124 Generation of free radicals 116 126.96.36.199 Propagation of radical damage in collagen 118 6.4 Conclusion 121 7. REFERENCES 123 8. APPENDIX 133
ix LIST OF FIGURES
FIGURE 1-1 The golden spider major ampullate gland 5 FIGURE 1-2 Silk glands, silk types and silk uses of Nephila clavipes 7 FIGURE 1-3 Nephila clavipes MaSp1 repeating units 9 FIGURE 1-4 Consensus amino acid sequence of minor ampullate silk protein 1 and 2 from N. clavipes 11 FIGURE 1-5 Computer models of the polyAla and polyGly/Ala segments 17 FIGURE 1-6 Computer model of the GGX repeat region 20 FIGURE 1-7 Computer model of a silk β-spiral (GPGGSGGPGGY) 21 FIGURE 2-1 (A) Theoretical phase diagram for polystyrene-polyisoprene diblock copolymers; (B) Ordered-state morphologies found in diblock copolymer melts 33 FIGURE 2-2 Structural hierarchies in block copolymers 37 FIGURE 3-1 The protein sequences of spider silk-like block copolymers 46 FIGURE 3-2 Overview of Recombinant DNA Technology 52 FIGURE 3-3 DNA sequences of synthetic spider silk blocks (A, B) and the pET30L adaptor sequence (C) 54 FIGURE 3-4 pET30a(+) vector map 55 FIGURE 3-5 Cloning strategy of spider silk-like block copolymers 56 FIGURE 3-6 SDS-PAGE of expressed spider silk-like block copolymers stained with Colloidal Blue 58 FIGURE 3-7 Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectra: (A) BA block copolymer, (B) BA2 block copolymer 59 FIGURE 4-1 FTIR spectra of silk-like block copolymers 66 FIGURE 4-2 Molecular assemblies of spider silk-like block copolymers in water and isopropanol 70 FIGURE 4-3 Morphology (A) and anatomy (B and C) of the BA6 spider silk block copolymer 72 FIGURE 4-4 AFM images of BA3, BA2, and AB3 block copolymers in
x isopropanol 76 FIGURE 4-5 Partial phase diagram of spider silk block copolymers at a constant concentration 77 FIGURE 5-1 pET30LF adaptor sequence and silk PCR primers 89 FIGURE 5-2 2% gel electrophoresis of the PCR products 89 FIGURE 5-3 Silk Block Copolymers Stained with Zinc Stain 92 FIGURE 5-4 SDS-PAGE of histidine free spider silk block copolymers stained with reversible Zinc (A) and Colloidal Blue (B) Stains 92 FIGURE 6-1 The components of collagen FTIR spectra (2800-3500 cm -1 region) 101 FIGURE 6-2 Types of water bridges in collagen 105 FIGURE 6-3 (A) The deconvolved amide I and amide II absorbance bands of native collagen films; (B) The deconvolved amide I and amide II absorbance bands of heat-denatured collagen films; (C) Comparison of amide I and amide II absorption bands of native and heat-denatured collagen films 106 FIGURE 6-4 A) SDS tris-glycine gel of native (NC) collagen type I stained with SilverXpress before and after UV254nm irradiation; (B) SDS bis-tris gel of native (NC) collagen type I stained with SimplyBlue; (C) SDS tris-glycine gel of heat-denatured collagen type I stained with SilverXpress before and after UV254 nm irradiation 112 FIGURE 6-5 AFM topology (A,C) and phase (B,D) images of native (top) and heat-denatured (bottom) collagen films before treatment with UV254nm 114 FIGURE 6-6 Potential mechanism of collagen degradation by UV-254 116 FIGURE 6-7 Potential mechanisms of collagen backbone cleavage 121
xi LIST OF TABLES TABLE 1-1 Comparison of the ensemble repeats and core units in spider silk proteins 15 TABLE 1-2 Comparison of mechanical properties of spider silks 23 TABLE 2-1 Sequences of diblock and triblock elastin-like protein copolymers 40 TABLE 3-1 Molecular characteristics of spider silk-like block copolymers 47 TABLE 3-2 Protein yields and molecular weight of spider silk-like block copolymers 57 TABLE 3-3 Predicted and observed molecular weight of spider silk block copolymers 60 TABLE 4-1 The peak position, intensity and band assignment of spider silk block copolymers 67 TABLE 4-2 Changes in random coil and β-sheet contents of the spider silk block copolymers 69 TABLE 4-3 Observed and predicted morphological characteristics of spider silk block copolymers 79 TABLE 6-1 Changes in the frequencies of bands contributing to the 2800-3500 cm -1 region 102 TABLE 6-2 Assignment of bands of the 2800-3500 cm -1 region 102 TABLE 6-3 Changes in the relative integral absorbance of the amide I band components of native collagen (NC) and heat-denatured collagen (DC) 107 TABLE 6-4 Assignment of the band components of the amide I region 109 TABLE 6-5 Assignment of the band components of the amide II region 110 TABLE 6-6 Location, percent area contribution and standard deviation of the amide II components before and after exposure to UV radiation 111 TABLE 6-7 Dimensional changes in the scratching lines on the collagen films before and after exposure to UV-254 nm 115
I am sincerely grateful to my advisor Professor David Kaplan for believing in me as a scientist and providing support, encouragement and a great working environment throughout this study. I know that you are the best mentor and teacher and I was lucky to be your student!
I also gratefully acknowledge my co-advisor Professor Peggy Cebe for her critical comments, suggestions and all that time she spent with me discussing this and other studies. I appreciate your wisdom, knowledge and kindness!
I thank all my committee members: Prof. Elena Rybak-Akimova, Prof. Krishna Kumar, Prof. Marc d’Alarcao, and Dr. Rajesh Naik for taking time meeting with me and helping me to become a professional researcher.
I am grateful to Tufts University for finding real friends such as Heather, Hania, Aneta, Dean, Monica and Silvia. Thank you all for being with me and putting a positive spin on things during our discussions in the lab or at a party. I know that you are my friends forever and ever. I am grateful to Tufts University for having my wedding here with all my friends and professors and I believe Tufts is my second home!
I thank to all my undergraduate students working with me: Julia, Matt, Will, William, Shannon, Ted and Jack as well as Shreevidhya for taking over my project and putting things together.
I am thankful to Carmen, Amanda, Mike C., Mike L., Mike M., Vikas, Kiran, Xiaohui, Subu, Deniz, Jen, John, Sara, Guokui, Keleigh and many others for your smiles, conversations, encouragements and exceptional friendly environment I have being working in. You are all great and I wish you all the best!
Above all, I would like to thank my mother, father and Zhena for all the love and support they have given to me throughout all my years in school. Without you I would not be able to do it! You bought me up and you gave me everything to become a good person!
And finally, I am thankful to Artem, my husband and my love. I appreciate your understanding, encouragement and wonderful support provided to me during these years. I want to dedicate this dissertation to you, just to show how much I love you and how much I miss you now! Even though we are apart right now, I know that we are going to be together again and this will happen very soon! I love you and I am ready to spend all my life with you! Lena Tokareva (Rabotyagova) July 19, 2008 Ukraine
1 PART I: SPIDER SILK BLOCK COPOLYMERS
Project Overview The natural processes of self-assembly have been at work since the creation of the Universe. In modern times scientists attempt to understand and find ways to utilize the self-assembly processes for human needs. Nature’s ability to assemble such a variety of molecules into biofunctional architectures inspires scientists to mimic Nature’s design in producing biological materials with desired functions. In our research, we focus on spider silk as a biopolymer, which offers the unique combination of high elasticity, toughness, and mechanical strength along with biological compatibility and biodegrability. We consider spider silk as an example of a natural block copolymer, in which hydrophobic and hydrophilic blocks are linked together generating a functional polymer with extraordinary properties. Since silks resemble synthetic block copolymer systems, we adopt principles of block copolymer design from the synthetic polymer literature (e.g. block design, phase diagram) to build a native block copolymer system based on a spider silk sequence. Moreover, we consider spider silk to be a useful model to study the relationship between structure and properties in our system. Our primary goal was to generate a new family of functional spider silk-like block copolymers to provide a fundamental insight into relationships between peptide sequence chemistry, block composition, and length and the observed morphological and structural features on the other side. This insight links knowledge from native block copolymer systems (e.g. protein chemistry) with an understanding of self-assembly behavior (e.g. morphology control) arising from synthetic block copolymer systems.
2 Aside from the fundamental perspective, we anticipate a number of potential applications for our biomaterials such as in controlled release devices, functional coatings and components of tissue regeneration materials. The specific aims for our study were the following: a) the formation of novel well-defined silk-based materials and b) control of morphological and structural features via precise block design. The model system utilizes protein sequences found in native spider dragline silk (Nephila clavipes); genetic variants of the major ampullate dragline silk protein I (MaSpI) were used to construct building blocks via genetic engineering to employ them in our design. The unique aspect of this study is the intrinsic ability of spider silks to form beta- sheet crystals that gave us an opportunity to generate the first protein based semicrystalline block copolymer system. We have successfully engineered spider silk- like block copolymers, and studied their secondary structure, morphological features and assembly behavior. These recombinant block copolymers have been used to develop a partial phase diagram to gain further knowledge towards the engineering of well-defined and predicable materials with precise control over primary sequence, length, and surface chemistry. In addition to this, our biomaterials can be utilized for a number of potential applications, such as biocompatible scaffolds and small molecules release devices. We anticipate that our model study can provide a foundation upon which future protein based block copolymer systems will be designed and implemented.
3 CHAPTER 1: SPIDER SILKS
1.1 Spider Silks - Ideas for Nanoscience The order Araneae (spiders) contains over 37,000 species that have ability to produce silk (Hu et al., 2006), a key protein for spiders to survive. Spiders use silk for a variety of practical purposes including: arresting a fall, swathing a prey, building a web, lining burrows, or making egg cases (Rising et al., 2005). Spider silk is a light weight material; it is an extremely strong and elastic material that exhibits mechanical properties comparable to the best synthetic fibers produced by modern technology (Gosline et al., 1999). In fact, studies have shown that on an equal weight basis, spider silk is stronger than steel and Kevlar (Gosline et al., 1999; Kluge et al., 2008). Silk fibers are protein-based biopolymer filaments or threads secreted by specialized abdominal glands connected to the spinnerets, ducts or spigots, and are used in different combinations to produce structures for prey capture, reproduction and locomotion (Hu et al., 2006). At least six different types of silk proteins are known (Lewis, 2006a). They differ in primary sequence, physical properties and functions (Hu et al., 2006). The complex spinning process allows spiders to transform soluble silk proteins into solid fibers using water as the solvent at ambient temperatures and pressures, giving rise to an environmentally safe, biodegradable, and high performance material. Diverse and unique biomechanical properties together with biocompatibility and slow rate of biodegradation make spider silks excellent candidates to be studied as biomaterials for tissue engineering (e.g. guided tissue repair and drug delivery), cosmetic products (e.g. nail and hair strengthener, skin care), industrial materials (e.g. nanowires,
4 nanofibers, surface coatings) (Vendrely and Scheibel, 2007). However, it is impossible to study spider silks as biomaterials without a complete understanding of the anatomy of spider glands, the silk spinning process, and the role of silk primary structural elements and their contribution to the physical and biological properties of the biopolymer.
1.1.1 Anatomy and Physiology of the Spider Spinning Apparatus (Nephila clavipes). Orb web spiders produce silk in seven morphologically distinct glands. However, it is thought that all spider glands evolved from a single type of gland (Vollrath and Knight, 2001) which diverged in anatomy, morphology and luminal content. The presence of different amino acids in the luminal content within each gland is responsible for the secretion of defined silk fibers with specific functions. Today, most research is focused on the major ampullate gland, which produces dragline silks. In this study, the major ampullate gland is used as a model system to describe the manufacturing of the silk fibers in Araneae. In Nephila clavipes glands are located within the abdomen of the spider and each type of gland occurs in pairs with bilateral symmetry (Lewis, 2006a).The major ampullate gland can be schematically divided into four zones as shown in Figure 1-1: the tail zone, responsible for synthesis and secretion of spider silk proteins, the lumen (also known as the sac), involved in protein accumulation, the spinning duct responsible for the alignment of silk fibers, and the spigot for final fiber production (Lewis, 2006b).
Figure 1-1. The golden spider major ampullate gland; photo of Jose Bico, modified from web.mit.edu/newsoffice/, scale bar is 200 µm.
The gland represents a reservoir of soluble silk proteins that are synthesized in specialized columnar epithelium cells in the tail zone and secreted into the lumen, where the proteins are stored as a highly concentrated liquid crystalline solution (Rising et al., 2005). According to Vollrath and Knight (Vollrath and Knight, 1999), the tail zone is named the A-zone and the first part of the lumen is named the B-zone, based on the presence of the single type of cells known as the tall columnar secretory epithelium. The spinning duct follows the lumen, which is folded into an S-shape and narrows to the end (Vollrath and Knight, 1999). Within this duct, silk proteins are present as liquid dope in which polymers are aligned to reach the optimal orientation for spinning (Rising et al., 2005). The spinning duct consists of three limbs. In each limb spidroin molecules have different orientation. In the first limb, silk proteins are anchored perpendicular to the cuticle lining but parallel to each other (Vollrath and Knight, 1999). In the second limb of the duct, silk molecules are bent in such way allowing the formation of layered discs Lumen (Sac) Tail Spinning duct Spigot Silk thread
6 made up the amphiphilic rod-shaped molecules that are still connected to the ducts cuticle lining. Finally, in the third limb the silk dope pulls away from the cuticle lining forming a draw down taper (Rising et al., 2005). At this moment, the liquid silk solution is converted into a solid thread surrounded by water and it is thought that β-sheet formation is initiated during this process by means of the rapid extension flow that pulls silk molecules close together and aligns them using hydrogen bonding into β-sheets (Knight et al., 2000; Rising et al., 2006). During the silk dope journey through the spinning duct, a number of changes in the chemical environment take place: these include the lowing of pH from 6.9 in the first limb to 6.3 in the third limb; decreasing the sodium and chloride concentrations, and increasing of potassium, phosphate, and sulphate concentration. The increase in the concentration of potassium can be explained by the ability of potassium ions to convert structural water into bulk water. At the same time, the drop in pH together with ionic changes can facilitate the neutralization of repulsive negative charges aiding silk molecule alignment into β-sheets. The final part of the major ampullate gland is represented by the valve and the spigot. The valve is used as a clamping device to control spider dragline and as a pump to remove threads that are broken inside the spigot. The spigot (spinneret) is located at the very end of the gland from which the spider thread is drawn. It has been proposed that spigot is also capable of further modifying the silk thread (Rising et al., 2005). When silk protein is drawn from the spinning duct all the way down to the spigot, silk becomes progressively more dehydrated, slightly acidified, and birefringent suggesting that the orientation and shape of silk molecules have been already programmed.
7 1.1.2 Spider Silk Types and Their Modules One spider is capable of producing up to seven different types of silks with varying mechanical properties. Silks are produced in specialized glands including: the major and minor ampullate, tubuliform, flagelliform, aggregate, pyriform, and aciniform glands (Lewis, 2006b). Figure 1-2 depicts silk glands and silk types.
Figure 1-2. Silk glands, silk types and silk uses of Nephila clavipes; the figure is adopted from Vollrath and Porter, 2006 and reprinted with permission of The Royal Society of Chemistry.
One of the main goals of this section is to make the reader familiar with silk proteins, so the well-characterized silks, in particular major ampullate, minor ampullate Major Ampullate Silk Minor Ampullate Silk Aciniform Silk Flagelliform Silk Tubuliform/CylindriformSilk
8 and flagelliform silks and recently discovered silk types such as aciniform, tubuliform, fiber coating, and pyriform silks will be discussed.
188.8.131.52 Major Ampullate Silk Proteins The golden orb weaver spider, Nephila clavipes, produces dragline silk in the major ampullate gland (Vollrath and Knight, 2001). It is composed of at least two different proteins that are held together by three to five disulphide bonds (Knight and Vollrath, 2001). The protein complex has a molecular weight of 320kDa and composed of major ampullate dragline silk protein 1 (MaSp1) and major ampullate dragline silk protein 2 (MaSp2). In 1990 Hu and Lewis screened the cDNA library with a probe based on a short peptide isolated from acid hydrolyzed N. clavipes dragline silk and found the gene encoding MaSp1 (Xu and Lewis, 1990). The total length of MaSp1 transcript is estimated to be 12.5 kb (Hayashi et al., 1999). Analysis of the primary sequences of MaSp1 from different species reveled regular small peptide motifs that can be grouped into three categories: poly-alanine region (GA/An), glycine-rich region (GGX), and a non-repetitive N- and C-terminus (Hu et al., 2006). Additional studies demonstrated that poly-alanine regions can contain between four to seven alanine residues and the X position in the GGX repeat can be occupied only by Y, L or Q (Hayashi et al., 1999). Figure 1-3 depicts the repeating protein units of MaSp1 revealed by Hu and Lewis (Xu and Lewis, 1990).
Figure 1-3. Nephila clavipes MaSp1 repeating units (adopted from Xu and Lewis, 1990).
MaSp2 possesses similar structural motifs: poly-alanine region (GA/A n ), GPGGX region and a non-repetitive N-terminus and C-terminus. The main difference between MaSp1 and MaSp2 is the presence of proline residues accounting for 15% of the total amino acid content in MaSp2 (Hu et al., 2006). To make it clear, MaSp1 is a proline free protein. By calculating the number of proline residues in N. clavipes dragline silk, it is possible to estimate the fiber content - 81% MaSp1 and 19% MaSp2 (Brooks, 2005). Different spiders have different rations of MaSp1 and MaSp2. For example, a dragline silk fiber from the orb weaver Argiope aurantia contains 41% MaSp1 and 59% MaSp2 (Huemmerich et al., 2004). Such changes in the ratios of major ampullate silks can dictate the performance of the silk fiber (Vollrath and Knight 1999). --------QGAGAAAAAA-GGAGQGGYGGLGGQG ---------------------AGQGGYGGLGGQG ------AGQGAGAAAAAAAGGAGQGGYGGLGSQG AGR---GGQGAGAAAAAA-GGAGQGGYGGLGSQG AGRGGLGGQGAGAAAAAAAGGAGQGGYGGLGNQG AGR---GGQ--GAAAAAA-GGAGQGGYGGLGSQG AGRGGLGGQ-AGAAAAAA-GGAGQGGYGGLGGQG ---------------------AGQGGYGGLGSQG AGRGGLGGQGAGAAAAAAAGGAGQ---GGLGGQG ------AGQGAGASAAAA-GGAGQGGYGGLGSQG AGR---GGEGAGAAAAAA-GGAGQGGYGGLGGQG ---------------------AGQGGYGGLGSQG AGRGGLGGQGAGAAAA---GGAGQ---GGLGGQG ------AGQGAGAAAAAA-GGAGQGGYGGLGSQG AGRGGLGGQGAGAVAAAAAGGAGQGGYGGLGSQG AGR---GGQGAGAAAAAA-GGAGQRGYGGLGNQG AGRGGLGGQGAGAAAAAAAGGAGQGGYGGLGNQG AGR---GGQ--GAAAAA--GGAGQGGYGGLGSQG AGR---GGQGAGAAAAAA-VGAGQEGIR---GQG ---------------------AGQGGYGGLGSQG SGRGGLGGQGAGAAAAAA-GGAGQ---GGLGGQG ------AGQGAGAAAAAA-GGVRQGGYGGLGSQG AGR---GGQGAGAAAAAA-GGAGQGGYGGLGGQG VGRGGLGGQGAGAAAA---GGAGQGGYGGV-GSG ----------ASAASAAAASRLSS
10 In addition to the repetitive units, dragline silks contain non-repetitive N and C- termini (Hu and Lewis, 1990; Bini et al., 2004; Lewis, 2006a). The length and amino acid composition of the C-terminus have been shown to be conserved between major ampullate proteins from different spiders (Ittah et al., 2006). It has been proven that the C-terminal region is extremely important for solubilizing silk proteins in the highly concentrated spinning dope (Hu et al., 2006). In contrast to the C-terminus, very little is known about the N-terminal region of spider silks. It is only known that the sequence possesses an enzymatic cleavage site and has usual amino acid composition and may have a signal peptide (Lewis, 2006b; Rising et al., 2006). Further studies are needed to determine a role for the N-terminal region in silk proteins.
184.108.40.206 Minor Ampullate Silk Proteins The minor ampullate silk (N. clavipes) consists of two distinct proteins, the minor ampullate dragline silk 1 and silk 2 (MiSp1 and MiSp2), with transcripts of 9.5 and 7.5 kb, respectively, generating proteins of 320kDa and 250 kDa in size (Hayashi et al., 1999). The cDNA sequence representing the MiSp1 transcript from N. clavipes was published shortly after the initial discovery of the MaSp1 sequence (Colgin and Lewis, 1998). Figure 1-4 shows the consensus repeats found in MiSp1 and MiSp2 of N. clavipes.
Figure 1-4. Consensus amino acid sequence of minor ampullate silk protein 1 and 2 from N. clavipes as revealed by Colgin and Lewis (1998).
The minor ampullate dragline silk proteins possess similar structural modules to the major ampullate silk proteins; however, there are some differences. The similarities include the presence of GGX and poly(A) modules. However, poly(A) modules of the MaSps are replaced by poly(GA) repeats. The organization of modules is also similar to MaSps; whereas, the number of repeats is different (Lewis, 2006b). Another difference is the presence in MiSps of a conserved serine-rich non-repetitive 137 amino acid sequence that is termed a spacer region (Colgin and Lewis, 1998). The spacer regions have a similar serine composition relative to the amorphous regions of Bombyx mori and are highly conserved among different spider species. It is hypothesized that the spacer plays a role in fiber formation by serving as a site of protein-protein interactions (Hu et al, 2006). The functional role of the spacer remains to be determined.
220.127.116.11 Flagelliform Silk Proteins The major component of the capture spiral of a spider web is flagelliform silk (Flag), which is produced by the flagelliform gland. Hayashi and Lewis retrieved a partial cDNA sequence of flagelliform silk from N. clavipes flagelliform gland (Hayashi and Lewis, 1998). It was found that the flagelliform gene encodes an mRNA transcript of 15 [GAGGAGGYGRGAGAGAGAAAGAGAGAGGYGGQGGYGAGAGAGAAAAAGA] n SPACER [GAGVGAAAAGFAAGAGGAGGYR] n SPACER MiSp1 MiSp2
12 kb that generates a protein of 500 kDa (Hu et al., 2006). Flagelliform silk has several distinctive features: a) possesses GPGGX module; b) has a highly conserved spacer with charged and hydrophilic amino acids; and c) possesses a nonrepetitive C-terminal region (Lewis, 2006b). Such structural combination gives flagelliform silk incredible elasticity – it can stretch up to 200% of its own length. The GPGGX motive is thought to be an explanation of the unique elasticity of flagelliform silk. As one remembers, the GPGGX motive is also found in MaSp2. Flagelliform silk has the same GPGGX repeat but it occurs seven times more in flagelliform than in MaSp2. Thus, flagelliform silk exhibits seven times more elasticity than MaSp 2 (Lewis, 2006a). The molecular mechanism of such great elasticity is the formation of a molecular spring (β-spiral), in which a series of type II β-turns are linked together. The spacer region has no homology with any known proteins and its functions are unknown. Also unknown is the function of the C-terminal region, which shows no similarity to the C-termini of MaSps and MiSps (Lewis, 2006a; Becker et al., 2003).