Relating the Structure of Insect Silk Proteins to Function
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Relating the structure of insect silk proteins to function Andrew A. Walker April 2013 A thesis submitted for the degree of Doctor of Philosophy of the Australian National University For Lucy Declaration This thesis represents my own original research work, and has not been submitted previously for a degree at any university. To the best of my knowledge and belief this thesis contains no material previously published or written by another person, except where due reference is made. One of the realities of doing research in a modern laboratory is the necessity of working closely with other members of a research team and external collaborators. For this reason some of the experimental results presented in this document were obtained by people other than myself. They are presented here to maintain a coherent narrative. A comprehensive list of these instances follows: liquid chromatography-mass spectrometry results in chapters 3, 4 and 6, and some of those in chapter 5, were obtained by Sarah Weisman; all Raman scattering spectra were obtained by Jeffrey S. Church; in chapters 4 and 6, I make use of a cDNA library constructed by Holly Trueman; all nuclear magnetic resonance spectra were obtained by Tsunenori Kameda; all amino acid analyses are results obtained by a commercial service at the Australian Proteome Analysis Facility. Andrew Walker April, 2013 Canberra, Australia Acknowledgements I would first like to thank my principal supervisor Tara Sutherland, who is the sort of person who can look at a lawn and see all the four-leaf clovers. She has taught me much about protein science but more about good management, good writing, happy workplaces, and how to publish. My supervisors at ANU, comprising one John (Trueman) and two Pauls (Cooper and Carr), have been gentle and constructive in their criticism, creative and bold in their thinking, and I thank them. I would like to thank each member of the silks group at CSIRO Ecosystem Sciences whom I have been privileged to work with in the course of this project: Sarah Weisman, Holly Trueman, Sri Sriskantha, Peter Campbell, Michelle Williams, and Ros Mourant. I would like to thank the other individuals with whom we have worked over the course of this project: David Merritt, Jeff Church, Tsunenori Kameda, Andrea Woodhead, and John Ramshaw and his team. I would like to thank everyone who has provided me with feedback regarding passages of this thesis including Roger Jones, Holly Trueman, Dek Woolfson, Shoko Okada, Mary Walker, Cameron Ewens, and Ashley Walsh. Thanks go to Ros Mourant, David Rentz, You Ning Su, Alison Rowell and John Trueman for advice and assistance in collecting raspy crickets; to the long list of people who helped collect silverfish and praying mantises; to Paul Cooper and Eric Hines for help with dissection and photography; and to Sri Sriskantha, Michelle Williams, Peter Campbell and Xiaoyi Wang for assistance with laboratory procedures. I am grateful to the ANU and CSIRO for supporting this project financially. I would also vii like to thank the Australian Proteomic Analysis Facility and the Australian Synchrotron. Lastly and most importantly, my thanks go to my wife Nicole, my mother Lin and fa- ther Ian, my sister Mary and brother Graham, and my friends. They have provided me with overwhelming love and support and I could not have performed this work without them. viii Abstract Silks are extracorporeal fibrous protein materials. Classically, silkworm (Bombyx mori) and orb-spiders (Arachnida: Araneidae) have served as model organisms in which to investigate silk protein structure-function relationships. However, silk production has evolved multiple times in insects. The silk proteins of many insects do not fold into the β-sheet structures found in silkworm and spider silks but into coiled-coils, collagen helices or polyglycine helices. Therefore, the structure-function relationships elucidated for silkworm and spider silk proteins may be too narrow to apply to insect silk proteins generally. To increase the available data, I examined silk production by raspy crickets (Orthoptera: Gryllacrididae), silverfish (order Thysanura), praying mantises (order Mantodea), glow-worms (Diptera: Keroplatidae), and sawflies (Hymenoptera: Tenthredinidae). Silk protein primary structures were investigated using transcriptomics, mass spectrometry, and amino acid analy- sis; secondary and tertiary structures were investigated by infrared and Raman spectroscopy, nuclear magnetic resonance, circular dichroism spectroscopy, and bioinformatics. Novel features of silk production were related to idiosyncrasies of each insect group, while features found in multiple silk-producing groups were associated with general mechanisms of silk production. A comparative analysis of silk proteins revealed a correlation between predominant secondary structure type and more general architectural features such as length and repeat regularity: silk proteins that fold into coiled-coils and collagen helices had low molecular weights and high repeat regularity, suggesting they fold into short semi-rigid rods; β-sheet-forming silk ix proteins were found to be more variable in molecular weight and have lower repeat regularity. Based on these data, I propose three major mechanisms of silk fabrication by insects: a) mesogenic ordering of short rod-like proteins, a process for which the coiled-coil and collagen structures are well-suited; b) molecular extension of long flexible protein chains to promote intermolecular bonding, which is suitable for the formation of β-sheet-rich silks; and c) entan- glement of protein chains, which is suited to silks with a high degree of disorder. Thus, many features of insect silk proteins are adaptations for material fabrication. In a few cases, particular structural motifs constituted adaptations conferring a mechanical property required for the silk's function in the solid state. However more often proteins were observed to have features promoting dense protein packing in a general way. I explain these data by consideration of how silk mechanical behaviour relates to the fitness advantage conferred to individual insects by silk production. Specifically, I suggest protein features ensuring struc- tural homogeneity and molecular orientation result in silk materials with mechanical properties sufficient for most purposes. Further increases in properties such as strength lead to little or no fitness increase. Local maxima in the fitness landscape associated with distinct protein secondary structures or fabrication mechanisms trap silk proteins in one of several states. Over- all, silk protein evolution can to a large extent be understood as convergence of a number of independently co-opted proteins of other functions toward one of several distinct functional archetypes. x Contents Acknowledgements vii Abstract ix 1 Introduction 1 1.1 What is a silk? . 2 1.2 Silk-producing insects . 3 1.3 Silk glands . 5 1.4 Silk molecular structure . 12 1.5 Silks with extended-β-sheet crystallites . 15 1.6 Silks with cross-β-sheet crystallites . 17 1.7 Silks with coiled-coil crystallites . 18 1.8 Silks with collagen and polyglycine II crystallites . 19 1.9 Silk fabrication . 19 1.10 Silk mechanical behaviour . 23 1.11 A comparative approach to understanding silk . 26 2 Silk from crickets: A new twist on spinning 27 2.1 Abstract . 29 2.2 Introduction . 30 xi CONTENTS 2.3 Results . 32 2.3.1 Raspy cricket build shelters by using silk fibres and films to join other materials . 32 2.3.2 Fibres and films have a similar molecular structure . 34 2.3.3 Silk is produced from acinar labial glands . 36 2.3.4 Raspy cricket silk proteins . 39 2.4 Discussion . 43 2.5 Materials and Methods . 48 2.5.1 Insects . 48 2.5.2 Microscopy . 48 2.5.3 Raman Spectroscopy . 49 2.5.4 X-ray scattering . 49 2.5.5 cDNA library construction . 50 2.5.6 Mass spectrometry . 50 3 Silverfish silk is formed by entanglement of randomly coiled protein chains 53 3.1 Abstract . 54 3.2 Introduction . 55 3.3 Results . 58 3.3.1 Silverfish produce very fine non-birefringent silk threads . 58 3.3.2 Silverfish silk has low chemical stability and is made from high molecular weight proteins . 58 3.3.3 Silverfish silk consists of randomly coiled proteins . 60 3.3.4 Amino acid composition of silverfish silk . 70 3.4 Discussion . 73 3.5 Materials and methods . 78 3.5.1 Insects and silk collection . 78 xii CONTENTS 3.5.2 Microscopy . 78 3.5.3 Solubilisation and electrophoresis . 78 3.5.4 Raman and FTIR spectroscopy . 79 3.5.5 Amino acid composition . 80 4 Natural templates for coiled-coil biomaterials from praying mantis egg-cases 81 4.1 Abstract . 83 4.2 Introduction . 84 4.3 Materials and methods . 88 4.3.1 Insects and dissections . 88 4.3.2 Fourier transform infrared spectroscopy . 88 4.3.3 Solid-state nuclear magnetic resonance . 88 4.3.4 Construction of cDNA libraries . 89 4.3.5 Mass spectrometry . 89 4.3.6 Sequence analysis . 90 4.3.7 Recombinant protein expression and purification . 90 4.3.8 Circular dichroism . 91 4.3.9 Fabrication of solid protein materials . 92 4.4 Results . 93 4.4.1 Oothecae are composed of low molecular weight proteins folded into coiled-coils . 93 4.4.2 Two main structural proteins identified by LC-MS . 95 4.4.3 Mantis fibroins form coiled-coils with an unusual alanine/aromatic core . 99 4.4.4 Recombinant fibroins form coiled-coils in solution and solids . 102 4.5 Discussion . 106 4.6 Conclusions . 109 xiii CONTENTS 5 Molecular mechanisms enabling prey capture by glow-worm silk fibres 111 5.1 Abstract . 112 5.2 Introduction . 113 5.3 Results . 117 5.4 Glow-worm snares are a composite of silk and mucus . 117 5.4.1 Crystallites in glow-worm silk have the cross-β-sheet structure . 117 5.4.2 Glow-worm silk contains long hydrophilic proteins protease inhibitors .