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The Role the N-Terminal Domain Plays in Spidroin Assembly Krystal Audrey Cadle Clemson University, [email protected]

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The Role the N-Terminal Domain Plays in Spidroin Assembly Krystal Audrey Cadle Clemson University, Kcadle@G.Clemson.Edu Clemson University TigerPrints All Dissertations Dissertations 8-2016 The Role the N-terminal Domain Plays in Spidroin Assembly Krystal Audrey Cadle Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Genetics and Genomics Commons Recommended Citation Cadle, Krystal Audrey, "The Role the N-terminal Domain Plays in Spidroin Assembly" (2016). All Dissertations. 2296. https://tigerprints.clemson.edu/all_dissertations/2296 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]. THE ROLE THE N-TERMINAL DOMAIN PLAYS IN SPIDROIN ASSEMBLY A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Genetics by Krystal Audrey Cadle August 2016 Accepted by: Dr. William Marcotte, Committee Chair Dr. James Morris Dr. Michael Sehorn Dr. Kerry Smith ABSTRACT One of the most interesting biomaterials known to man is spider silk. These fibers, composed primarily of protein, show beneficial mechanical and biological properties that have many uses in medical and industrial fields. Spider silk proteins (spidroins) are composed of three core components that consists of a C-terminal domain (CTD), repeat domain (R), and N-terminal domain (NTD). The NTD and CTD are highly conserved and are believed to form “multimeric strands” that make up spider silk. Although it is known that the NTD undergoes a conformational change that results in stable homodimer formation at acidic pH , the way in which the NTD contributes to fiber assembly is still under investigation. Here we provide label-free, real-time dimerization data of the wild-type major ampullate spidroin 1 (MaSp1) NTD from Nephila clavipes using the Octet RED96 system. This data shows over 25-fold increase in the affinity of wild-type NTD monomers at pH 5.5 compared to pH 7.0. These results are in agreement with previous work from our lab and others that show an increase in homodimer stability at acidic pH. This information correlates with the conformational change seen as the wild-type NTD transitions from a neutral to acidic pH environment. We found that NTD variants D45K and E84K abolished the conformational change that occurs at pH 5.5 and decreased dimer stability compared to wild-type MaSp1 NTD. We also found showed that variant K70D and double variant D45K/K70D exist in an intermediate conformation between that seen at pH 5.5 and pH 7.0 for wild-type NTD. This new information provides additional evidence to support the idea that a conformational change is directly connected ii to dimerization. Our data provides additional insight into which residues are necessary for this conformational change and subsequent dimerization, which allows for a better understanding of the homodimerization process. We also demonstrate production of MaSp2 “mini spidroins” in Saccharomyces cerevisiae. These “mini spidroins” contained a native CTD and NTD and eight repeat units and preliminary results show that glucose and raffinose preinduction media result in better protein expression using 24 hr and 72hr induction times, respectively. iii DEDICATION For my friends and family who have been so supportive. iv ACKNOWLEDGMENTS First I would like to thank Dr. William Marcotte, Jr. for allowing me to join his lab, and guiding me and supporting me throughout my graduate career. I would also like to thank my committee members Dr. James Morris, Dr. Michael Sehorn, and Dr. Kerry Smith for their suggestions and help. This work could not have been accomplished without the invaluable help of my past and current lab members Dr. Will Gaines, Dr. Todd Lyda, Dr. Congyue (Annie) Peng, Kelly McQueeney, Mikayla Spitler, Breanna Burks and Allison Nelson. v TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii DEDICATION ................................................................................................................ iv ACKNOWLEDGMENTS ............................................................................................... v LIST OF TABLES .......................................................................................................... ix LIST OF FIGURES ......................................................................................................... x CHAPTER I. LITERATURE REVIEW .............................................................................. 1 Types and Properties of Spider Silks ....................................................... 1 Major Ampullate Silk’s Components ...................................................... 7 Fiber Assembly ...................................................................................... 12 Expression Systems ............................................................................... 20 Uses for Spider Silk ............................................................................... 27 References .............................................................................................. 34 II. TRYPTOPHAN FLOURESCENCE OF Nephila clavipes MAJOR AMPULLATE SPIDROIN 1 N-TERMINAL DOMAIN VARIANTS.......................... 45 Abstract .................................................................................................. 45 Introduction ............................................................................................ 46 Materials and Methods ........................................................................... 49 Results .................................................................................................... 52 Discussion .............................................................................................. 66 References .............................................................................................. 72 vi Table of Contents (Continued) Page III. OCTET OPTIMIZATION AND HOMODIMERIZATION OF WILD-TYPE N-TERMINAL DOMAIN FROM MAJOR AMPULLATE SPIDROIN 1 ................................................................. 75 Abstract .................................................................................................. 75 Introduction ............................................................................................ 76 Materials and Methods ........................................................................... 79 Results .................................................................................................... 86 Discussion .............................................................................................. 95 References .............................................................................................. 99 IV. DIMERIZATION OF Nephila clavipes MAJOR AMPULLATE SPIDROIN 1 N-TERMINAL DOMAIN ............................................................................................. 104 Abstract ................................................................................................ 104 Introduction .......................................................................................... 104 Materials and Methods ......................................................................... 108 Results .................................................................................................. 109 Discussion ............................................................................................ 114 References ............................................................................................ 121 V. PRODUCTION OF “MINI-SPIDROINS” IN Saccharomyces cerevisiae ................................................................... 125 Abstract ................................................................................................ 125 Introduction .......................................................................................... 125 Materials and Methods ......................................................................... 129 Results .................................................................................................. 132 Discussion ............................................................................................ 135 References ............................................................................................ 137 VI. CONCLUTIONS AND FUTURE DIRECTIONS .................................... 142 References ............................................................................................ 148 vii Table of Contents (Continued) Page APPENDICES ............................................................................................................. 150 A: Nephila clavipes Major Ampullate Spidroin1 N-terminal domain Tryptophan Fluorescence 320 nm / 338 nm Ratio Graphs ................................................................................................. 151 B: Permissions ................................................................................................ 154 Figure 1.1, Figure 1.3, Table 1.1 and part of Table 1.2 ....................... 155 Figure 1.2 and part of Table 1.2 ........................................................... 161
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