Identification and characterization of adhesive proteins in freshwater mussels towards the development of novel bioadhesives by Judith Ng A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Institute of Biomaterials and Biomedical Engineering University of Toronto c Copyright 2018 by Judith Ng Abstract Identification and characterization of adhesive proteins in freshwater mussels towards the development of novel bioadhesives Judith Ng Master of Applied Science Institute of Biomaterials and Biomedical Engineering University of Toronto 2018 The European freshwater mollusk Dreissena bugensis (quagga mussel) adheres to a variety of underwater surfaces via the byssus, a proteinaceous anchor, from which threads are secreted. The threads terminate in small, disc-shaped adhesive plaques which have adapted to turbu- lent conditions with remarkable adhesive strength for their size. Top adhesive candidates that demonstrated evidence for an adhesive role were identified via LC-MS/MS and relative quan- tification of proteins between the mussel plaque and adhesive interface. For the first time, a top candidate for adhesive activity (Dbfp7) was isolated directly from the quagga mussel. We aim to incorporate relevant protein motifs from these proteins into peptide mimics and to recombi- nantly express Dbfp7, leading to the development of novel bioadhesives for medical and dental applications. Understanding the molecular mechanisms and proteins responsible for adhesion in freshwater mussels will provide a paradigm for medical adhesives which function reliably on wet tissue surfaces with minimal inflammation. ii Acknowledgements There are several individuals whom I would like to thank. First and foremost, I would like thank my mentor and supervisor, Prof. Eli Sone. It has been an absolute joy to work under his tutelage for the past three years. His ability to empathize with students, to guide and encourage both their strengths and shortcomings, and to be present during every crest and trough of their project has made my personal journey as a graduate student in his group an unparalleled learning experience. I am very grateful to my committee members, Prof. Kevin Truong and Prof. Jonathan Roche- leau for their valuable feedback and thorough insight during the project. I would also like to thank Prof. Jayachandran Kizhakkedathu who welcomed me into a wonderfully productive and informative learning environment in his laboratory at UBC. I would like to thank all my colleagues in the Sone Lab who have been instrumental to the success of my project. Thank you to Magdalena Wojtas for her unique expertise in protein purification and for helping me every step of the way in experiments that have made almost an entire chapter of my thesis possible. Thank you to David Rees for his patience, diligence, and excellent research abilities that have made my transition to carrying forth his work well- facilitated and seamless. Endless thanks to Alex Lausch and Bryan Quan for always being willing to advise, troubleshoot, and along with all members of the Sone Lab, making the lab environment an incredible place to be. Thank you to Kenny Kimmins, Kate Li, Denise Yap, Bryan James, Minh-Tam Nguyen and Angelico Obille for their dedication and hours put into maintaining our well-oiled mussel aquarium system and for helping collect the mussels that have made this work possible. I would also like to acknowledge the labs that allowed access to their equipment and for their expertise, including Prof. Ben Ganss, Prof. Rocheleau, Prof. Radisic, Prof. Houry, and Prof. Master. I would also like to thank Paul Taylor of the SickKids Proteomics, Analytics, Robotics, & Chemical Biology Centre (SPARC BioCentre) for trypsin digestion, LC-MS/MS analysis and analysis guidance. Thank you to Reynaldo Interior for extensive amino acid analysis assistance and guidance. Last but not least, I would like to thank my family - my parents and my sister - for their unwavering support of all my goals, no matter how lofty, and for their constant and reassuring belief in my capabilities. The patience, love, and excitement they have always shown for my successes and lessons learned has made every hardship worthwhile. iii Contents Contents iv List of Tables vii List of Figures viii 1 Introduction 1 1.1 Background.......................................1 1.1.1 Mussel Biology and Adhesion.........................1 1.1.2 Marine Mussel Attachment..........................3 1.1.3 Recombinant and Peptide Mimic Experiments...............5 1.1.4 Animals as Models of Adhesion........................5 1.1.5 Mussels as Models for Adhesion........................6 1.1.6 Existing Freshwater Mussel Byssal Proteins.................7 1.2 Rationale and Motivation............................... 10 1.2.1 Limitations of Current Adhesives....................... 10 1.2.2 Weaknesses of Current Mussel-Inspired Adhesives.............. 11 1.2.3 Current Progress: Candidate Spatial Verification and Molecular Weight. 12 1.3 Project Objectives and Goals............................. 13 1.3.1 Objective 1: Identify quagga mussel adhesive protein candidates and se- lect the top candidate............................. 13 1.3.2 Objective 2: Purify and characterize the top quagga mussel adhesive protein candidate................................ 14 1.4 Document Overview.................................. 15 2 Quagga Mussel Adhesive Protein Identification 16 2.1 Abstract......................................... 16 2.2 Introduction....................................... 17 2.3 Methods......................................... 19 2.3.1 Mussel Thread/Plaque and Footprint Collection.............. 19 2.3.2 SDS-PAGE and Staining............................ 19 iv 2.3.3 Liquid Chromatography Tandem Mass Spectroscopy (LC-MS/MS).... 19 2.3.4 Protein Identification, Selection, and Analysis................ 20 2.4 Results & Discussion.................................. 20 2.4.1 Quagga Mussel Protein Distribution..................... 21 2.4.2 Determination of Putative Protein Roles................... 22 2.4.3 Byssal Proteins: All Sectors.......................... 24 2.4.4 Relative Protein Quantification of Footprint+Plaque Proteins....... 26 2.4.5 Relative Protein Quantification: Candidate Selection............ 28 2.4.6 Caveats of Experimental Design....................... 35 2.5 Conclusion....................................... 37 2.6 Acknowledgements................................... 37 2.A All Venn Diagram Sectors: Other Protein Roles................... 38 2.A.1 Structural Proteins............................... 38 2.A.2 Mucin-like Proteins............................... 38 2.A.3 Redox Proteins................................. 40 2.A.4 Mussel Proteins................................. 40 2.A.5 Protease Inhibitors............................... 41 2.A.6 Tyrosinase................................... 41 2.A.7 Cellular..................................... 42 2.B Relative Protein Quantification: Proteins of Interest................ 42 2.B.1 Rhinophore Proteins.............................. 42 2.B.2 Structural Proteins............................... 42 2.B.3 Mucus-like proteins............................... 43 2.B.4 Redox proteins................................. 43 2.B.5 Plasminogen-like Proteins........................... 44 2.C Justification for Targeted Footprint+Plaque Overlap Search............ 44 3 Dbfp7 Purification and Characterization 48 3.1 Abstract......................................... 48 3.2 Introduction....................................... 49 3.3 Methods......................................... 50 3.3.1 Protein Extraction............................... 50 3.3.2 Amino Acid Analysis.............................. 51 3.3.3 MALDI-TOF MS Spectrometry........................ 51 3.3.4 Liquid Chromatography Tandem Mass Spectroscopy (LC-MS/MS).... 51 3.3.5 Protein Identification, Selection, and Analysis................ 52 3.3.6 Circular Dichroism............................... 52 3.3.7 Quartz Crystal Microbalance......................... 52 3.3.8 Atomic Force Microscopy........................... 53 3.4 Results & Discussion.................................. 53 v 3.4.1 Phenol gland purification of mussel adhesive proteins............ 54 3.4.2 Seasonal Purification.............................. 55 3.4.3 Size and secondary structure of native Dbfp's................ 57 3.4.4 Adsorption studies of Dbfp's on SiO2, TiO2 and Au............ 62 3.5 Conclusion....................................... 67 3.6 Acknowledgements................................... 68 4 Discussion, Future Work & Conclusion 69 4.1 Future Work & Recommendations.......................... 69 4.1.1 Dbfp7 Structural Characterization Optimization.............. 69 4.1.2 Dbfp7 Surface-Sensitive Characterization Optimization.......... 71 4.1.3 Recombinant Expression of Dbfp7...................... 73 4.1.4 Expansion of Quagga Mussel Byssal Protein Knowledge.......... 73 4.2 Significance & Conclusions............................... 73 References 74 vi List of Tables 2.1 Quagga mussel Dbfp proteins found enriched and abundant at the adhesive in- terface........................................... 30 2.2 Large and abundant proteins identified by LC-MS/MS by Rees et al.[81] that did not originally meet acceptance criteria to be listed as a novel quagga mussel byssal protein, but were re-classified as potential byssal candidates due to the presence of Dopa within peptides. Bolded transcripts are both highly enriched and abundant at the footprint-plaque interface...................