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SPIDROIN N-TERMINAL DOMAIN: a PH SENSOR in the SPIDER SILK ASSEMBLY PROCESS William Gaines Clemson University, [email protected]

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SPIDROIN N-TERMINAL DOMAIN: a PH SENSOR in the SPIDER SILK ASSEMBLY PROCESS William Gaines Clemson University, Wagaine@Clemson.Edu Clemson University TigerPrints All Dissertations Dissertations 12-2010 SPIDROIN N-TERMINAL DOMAIN: A PH SENSOR IN THE SPIDER SILK ASSEMBLY PROCESS William Gaines Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Molecular Biology Commons Recommended Citation Gaines, William, "SPIDROIN N-TERMINAL DOMAIN: A PH SENSOR IN THE SPIDER SILK ASSEMBLY PROCESS" (2010). All Dissertations. 655. https://tigerprints.clemson.edu/all_dissertations/655 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. SPIDROIN N-TERMINAL DOMAIN: A PH SENSOR IN THE SPIDER SILK ASSEMBLY PROCESS A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Genetics by William Arthur Gaines, IV December, 2010 Accepted by: William R. Marcotte Jr., Committee Chair Albert Abbott Vance Baird Michael Sehorn i ABSTRACT Spider silks are protein-based fibers with remarkable mechanical qualities. Perhaps even more impressive is the spinning process in which the spider silk proteins (spidroins) are assembled from a highly soluble storage state into a well-ordered and insoluble fiber. Indeed, the ordered arrangement of spidroins, which is endowed by the spinning process, is the basis of fiber strength. However, the forces driving fiber assembly and the mechanisms by which spidroins respond those forces are only poorly understood. Spidroins have a tripartite domain architecture consisting of a large and repetitive central domain flanked by small, non-repetitive N- and C-terminal domains. Both terminal domains are well conserved among different types of spidroins, and they are believed to act as coordinators of spidroin assembly. This dissertation represents an important advancement of knowledge on the sequence, structure, and biochemical behavior of the highly conserved but greatly under studied N-terminal domain of major ampullate silks (MaSp-NTD). In my analysis of MaSp-NTD sequence, I demonstrated for the first time that one of the two types of MaSp genes (MaSp1) had been duplicated in the golden orb-weaving spider ( Nephila clavipes ) generating MaSp1A and MaSp1B genes. My publication of this work was contemporaneous with publications by research groups finding similar MaSp gene duplications in other spider species. Characterization of the RNA transcripts from MaSp1A and MaSp1B genes indicated that they are simultaneously co-expressed in adult ii female spiders. It also allowed experimental verification the transcription start sites in the MaSp genes. My biochemical analysis of three recombinant MaSp-NTDs indicate that they are highly pH responsive. Upon experiencing a mild acidification, as occurs in the spider silk gland, they undergo a dramatic change in tertiary conformation. Concurrent with the pH-dependent refolding, the interaction between MaSp-NTD molecules is strengthened resulting in considerably more stable MaSp-NTD homo-dimers. Through a collaboration with another research group, we are working to determine the structure of MaSp-NTD in both conformational states by NMR. By transducing the duct pH change into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD. iii DEDICATION For my two grandfathers, who have each been excellent role-models and have shown me what it means to embody greatness in the way they lived. iv ACKNOWLEDGMENTS Bill Marcotte - My mentor in science who was there at every step of the way with insightful guidance and encouragement. Mirko Hennig, - Our collaborators whose skill and enthusiasm have Stu Parnham, moved the project towards the structural answers of and Brendan Duggan ‘how’ that follow the biochemical discoveries of ‘what’. Mike Sehorn - His door has always been open to me and he has been extremely generous with his time in helping me decide on what experiments to do next and how to make sense of the experiments that I’ve just done. Bert Abbot - Thanks to our discussions I’ve gained a broader perspective on what silk is and what it can be. Vance Baird - An excellent teacher whose classes have taught me a great deal on the intricate nature of biological systems. Sarah Harcum - As both an engineer and a biologist, her perspective has been invaluable, especially when trouble-shooting. My family - Who have always been there for me. v TABLE OF CONTENTS Page TITLE PAGE....................................................................................................................i ABSTRACT.....................................................................................................................ii DEDICATION................................................................................................................iv ACKNOWLEDGMENTS ...............................................................................................v LIST OF TABLES........................................................................................................viii LIST OF FIGURES ........................................................................................................ix Chapter 1 - Literature review............................................................................................1 Types and Functions of Silk .........................................................................................1 Spider silks................................................................................................................1 Lepidopteran silks.....................................................................................................5 Other insect silks.......................................................................................................6 Molecular Constituents of Major Ampullate Silk.........................................................7 Core proteins.............................................................................................................7 Shell components....................................................................................................12 Non-protein components.........................................................................................13 Silk Genes: Structure and Evolution...........................................................................15 Tandem duplications in the repeat domain .............................................................16 Spidroin gene and domain duplications..................................................................20 Concerted evolution of the terminal domains.........................................................21 vi Table of Contents (Continued) Page The Fiber Production Pathway....................................................................................23 Major ampullate gland anatomy .............................................................................23 Pre-spun molecular organization ............................................................................27 Forces driving fiber assembly.................................................................................31 Fiber molecular organization ..................................................................................35 MaSp domain contributions to assembly................................................................39 The repetitive domain (MaSp-Rn).......................................................................40 The C-terminal domain (MaSp-CTD) ................................................................43 The N-terminal domain (MaSp-NTD)................................................................45 Efforts Towards Large-Scale Production of Spider Silks...........................................46 Expression and purification from biological hosts .................................................48 Synthetic analogs of silks........................................................................................50 Chapter 2 - Identification and characterization of multiple Spidroin 1 genes encoding major ampullate silk proteins in Nephila clavipes .................................................64 Chapter 3 - The Spidroin N-terminal Domain Promotes a pH-dependent association of silk proteins during self-assembly .........................................................................95 Chapter 4 - NMR Data Supporting Biochemical Findings...........................................134 Chapter 5 - NMR Assignment Note..............................................................................140 Chapter 6 - Conclusions and future directions..............................................................147 Appendix A - Supplemental figures .............................................................................154 vii LIST OF TABLES Table Page 1.1 The physical characteristics of silks and other materials.........................................3 1.2 The molecular constituents of spider silks ............................................................11 1.3 Summary of MaSp domain responsiveness ..........................................................40 2.1 MaSp1 N- and C-terminal domains present in seven N. clavipes
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