QUANTIFYING THE BIOMECHANICAL FORCES BETWEEN PROTEINS INVOLVED IN ELASTIN SYNTHESIS USING ATOMIC FORCE MICROSCOPY SEAN O’NEILL MOORE Bachelor of Science in Mechanical Engineering University of Notre Dame May 2015 Submitted in partial fulfillment of requirement for the degree MASTER OF SCIENCE IN BIOMEDICAL ENGINEERING at the CLEVELAND STATE UNIVERSITY December 2018 We hereby approve this thesis for SEAN O’NEILL MOORE Candidate for the Master of Science in Biomedical Engineering for the Department of Chemical and Biomedical Engineering and the CLEVELAND STATE UNIVERSITY’S College of Graduate Studies by Committee Chairperson, Dr. Chandrasekhar Kothapalli Department & Date Committee Member, Dr. Nolan Holland Department & Date Committee Member, Dr. Jason Halloran Department & Date Date of Defense: November 19, 2018 ACKNOWLEDGMENT First and foremost, I would like to thank Dr. Chandrasekhar Kothapalli for being my advisor throughout my biomedical engineering career at Cleveland State. I have learned many valuable skill sets and a more in depth appreciation for biomaterials and biomechanics under his guidance. This knowledge reaches far beyond that of teaching and I appreciate his insights. In addition, I would like to thank Dr. Nolan Holland of the Chemical and Biomedical Engineering Department, and Dr. Jason Halloran from the Mechanical Engineering Department for their invaluable advice and input on the project and taking the time to be a part of my thesis committee. Additionally, I would like to thank the Chemistry Department at Cleveland State as well as Dr. Holland’s Lab at Cleveland State for allowing me to utilize equipment necessary to run my experiments. I also want to thank the Baldock Lab at the University of Manchester in Manchester, England, for the generous gift of Fibulin-5. I would like to personally thank Gautam Mahajan in the Chemical and Biomedical Engineering Department at Cleveland State for his support and training of atomic force microscopy. Without his help and guidance during my time at Cleveland State, this project would not be possible. He has become a valuable friend in the process and I look forward to working with him in the future. Lastly, I would like to dedicate this work to my family, for whom none of this would be possible without their love and support QUANTIFYING THE BIOMECHANICAL FORCES BETWEEN PROTEINS INVOLVED IN ELASTIN SYNTHESIS USING ATOMIC FORCE MICROSCOPY SEAN O’NEILL MOORE ABSTRACT Elastogenesis is a complex and arduous process involving a series of biochemical and biomechanical interactions, from extraction of precursor proteins out of cells to cross-linking and deposition, ultimately forming elastic fibers. Elastin is an extracellular matrix protein comprised of two main proteins, tropoelastin and fibrillin-1, which gives tissues and organs resilience and elasticity under deformation. While the biochemical process by which elastic fibers are assembled is being elucidated, the forces between the constituents involved in this process remains unknown. To fill this knowledge gap in literature, this study measured and quantified the adhesive forces between elastic fiber components, primarily between tropoelastin, elastin binding protein (EBP), fibrillin-1, fibulin-5, and lysyl oxidase (LOX). The forces between other extracellular matrix proteins such as laminin-1, rat-tail collagen type I, and human collagen type I were also measured using atomic force microscopy (AFM). The interactions between an uncoated AFM cantilever tip with proteins (involved in elastin synthesis) coated on a cover glass showed that the adhesive forces increased with increasing molecular weight of the proteins. The adhesive forces between elastin synthesis proteins when tropoelastin was coated on the AFM cantilever tip revealed the strongest interaction to be when tropoelastin was coated on the cover glass (293.4 ± 20.5 pN), followed by LOXL2, fibulin-5, fibrillin-1, GLB-1, and mature elastin, which ranged between 100.3 pN to 199.8 pN. Introducing the cross-linker protein, LOXL2, decreased the adhesive force iv between the tropoelastin-coated AFM tip and tropoelastin-coated cover glass by roughly 100 pN. Lastly, the Worm-Like Chain (WLC) model provided insights into the bending rigidity, stiffness, and flexibility of elastin synthesis proteins as they were unfolding. This gives understanding to how each protein’s stretching capabilities under deformation contribute to elastin’s overall elastic nature. Knowing the forces between the individual molecules in elastogenesis could help understand their contributions to elastic matrix deposition and assembly, thereby the interactions between cells and extracellular matrix (ECM) components. This study led to bridging the gap between biochemical information and biomechanics, pertaining to elastic fiber assembly. v TABLE OF CONTENTS Page ABSTRACT .................................................................................................................... iv LIST OF TABLES .......................................................................................................... ix LIST OF FIGURES ..........................................................................................................x CHAPTERS I. INTRODUCTION ................................................................................................1 1.1 Elastin Overview ...........................................................................1 1.1.1 Major Structural Component ............................................1 1.1.2 Elastogenesis Matrix Synthesis.........................................4 1.1.3 Disease State .....................................................................7 1.1.4 Role of Individual Proteins in Elastic Fiber Formation ....9 1.2 Current Force Techniques and Limitations.................................13 1.3 Atomic Force Microscopy ..........................................................14 1.3.1 Biological Studies using Atomic Force Microscopy ......19 1.4 Lack of Biomechanics Research in Elastin Field .......................21 1.5 Research Aim and Project Goal ..................................................22 II. MATERIALS AND METHODS ........................................................................24 2.1 Coating AFM Tip with Tropoelastin, LOXL2, or Fibulin-5 ......24 2.2 Coating of Cover Glass with Tropoelastin, GLB-1, Fibulin-5, LOXL2, Fibrillin-1, Mature Human Aortic Elastin, Mouse Laminin-1, Rat-Tail Collagen-1, Human Collagen-1 .................25 2.3 Tropoelastin and LOXL2 Coating ..............................................26 2.4 Immunofluorescence Labeling of Cantilever Tip .......................27 vi 2.5 Fourier-Transform Infrared Spectroscopy Establishing Protein Coating on Cover Glass ..............................................................28 2.6 Contact Angle Establishing Protein Coating on Cover Glass .....29 2.7 AFM Experiments .......................................................................31 2.7.1 Force Curve Measurements ............................................33 2.7.2 Force Curve Interpretation ..............................................34 2.8 Single Protein Mechanics ...........................................................35 2.9 Hydrodynamic Radius Calculations ...........................................37 2.10 Statistical Significance ................................................................38 III. RESULTS ...........................................................................................................39 3.1 Contact Angle Measurements .....................................................39 3.2 FTIR Absorption .........................................................................40 3.3 Protein-Protein Adhesion Force ..................................................45 3.4 Single Protein Mechanics ...........................................................48 3.5 Hydrodynamic Radii for Folded Proteins ...................................51 IV. DISCUSSION .....................................................................................................53 4.1 Contact Angle Measurements .....................................................53 4.1.1 Elastogenesis Proteins Fibrillin-1, Fibulin-5, and GLB..53 4.1.2 Elastogenesis Proteins Tropoelastin and LOXL2 ...........54 4.1.3 Non-Elastogenesis Proteins ............................................55 4.2 FTIR Measurements....................................................................55 4.2.1 Fibrillin-1, Fibulin-5, and GLB-1 ...................................56 4.2.2 Tropoelastin and LOXL2 ................................................56 4.2.3 Non-Elastogenesis Proteins ............................................57 vii 4.3 Protein-Protein Biomechanical Interactions ...............................57 4.3.1 Non-Coated AFM Cantilever Tip Interactions ...............58 4.3.2 Tropoelastin-Coated AFM Tip Interaction with Fibrillin-1 and GLB-1 ......................................................................59 4.3.3 Tropoelastin-Coated AFM Tip with Tropoelastin in the Presence of LOXL2 ........................................................59 4.3.4 Tropoelastin-Coated AFM Tip with Fibrillin-1 in the Presence of LOXL2 ........................................................60 4.3.5 Increasing Dwell Time Effect on Adhesion Force .........61 4.3.6 Fibulin-5 Interaction with Elastogenesis Proteins ..........61 4.4 Tropoelastin and Fibrillin-1 Provide Elastin with Elasticity and Resilience ....................................................................................62