A REVIEW OF THE HIGHER-ORDER STRUCTURES AND APPLICATIONS OF COLLAGEN MIMETIC PEPTIDES A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Polymer Science Max Goldmeier May 2016 A REVIEW OF THE HIGHER-ORDER STRUCTURES AND APPLICATIONS OF COLLAGEN MIMETIC PEPTIDES Max Goldmeier Thesis Approved: Accepted: ______________________________ ______________________________ Faculty Advisor Dean of the College Dr. Nita Sahai Dr. Eric J. Amis ______________________________ ____________________________ Faculty Co-Advisor Dean of the Graduate School Dr. William J. Landis Dr. Chand Midha ______________________________ ____________________________ Department Chair Date Dr. Coleen Pugh ii ABSTRACT Collagen is a protein that is a major component of the human body and makes up thirty percent of the total protein in the human body. Collagen mimetic peptides (CMPs) have been used to study the formation of the triple helix of collagen and its higher- ordered structures. This article discusses how CMPs have been used to learn about the physical properties of the triple helix and how the amino acid sequence affects the physical properties, formation, and stability of the triple helix. CMPs have also been designed to form higher-ordered structures for various applications. This work reviews the design principles and strategies employed with various interactions to form higher- ordered structures from CMPs. These strategies include the use of electrostatic interactions, the incorporation of cysteine knots, and metal-induced self-assembly, among others. Many of these higher-ordered structures have applications such as biomolecule attraction, cellular adhesion, nanowire fabrication, and hydrogel formation. iii DEDICATION This work is dedicated to my family for their support and encouragement. iv ACKNOWLEDGEMENTS I would like to thank Dr. Nita Sahai and Dr. William Landis from the Department of Polymer Science at The University of Akron in Akron, OH for their guidance and support which made this work possible. v TABLE OF CONTENTS Page LIST OF TABLES…………………………………………………………………...…viii LIST OF FIGURES………………………..……………………......................................ix CHAPTER I. INTRODUCTION……………………………………………………..………………..1 1.1 Introduction………………………………………………...………………….1 1.2 Features of Collagen and Biosynthetic Process of Collagen Formation………5 II. HELICAL TWIST OF THE TRIPLE HELIX OF COLLAGEN…………………….10 III. TRIPLE HELIX STABILITY AND AMINO ACID SEQUENCE… ………….…..13 3.1 Introduction to Collagen Triple Helix Stability……………………………...13 3.2 Tertiary Amides……………………………………………………………...14 3.3 Stereoelectronic Effects……………………………………………………...15 3.4 Hydrogen Bonding…………………………………………………………...17 3.5 Electrostatic Interactions……………………………………………………..18 3.6 The Role of Glycine in Triple Helix Stability……………………………….19 3.7 Conclusion of Triple Helix Stabilizing Factors……………………………...21 IV. HIGHER-ORDER CMP STRUCTURES……………….............................………..22 4.1 Intro to Higher-Order Structure and Applications of CMPs…………………22 4.2 Pro-Hyp-Gly CMPs………………………………………………………….23 vi TABLE OF CONTENTS (CONT.) Page 4.3 Pro-Pro-Gly CMPs…………………………………………………………...24 4.4 CMP Amphiphiles…………………………………………………………...29 4.5 2+1 Strand Click Synthesis…………………………………………………..34 4.6 Cysteine Bridges……………………………………………………………..36 4.7 Aromatic Interactions………………………………………………………...38 4.8 Metal-Induced Self-Assembly……………………………………………….41 4.9 Electrostatic Interactions……………………………………………………..47 4.10 Nanodiamond-CMP Conjugates……………………………………………52 4.11 Polymerized-CMPs and CMP-Dendrimers…………………………………52 V. CONCLUSION ………………………………………………………………………59 VI. BIBLIOGRAPHY……………………………...…………………………………….61 vii LIST OF TABLES Table Page 1. Chart displaying the types of collagen, their classification, distribution, and pathology.2, 3………………………………………………………………….……2 2. The amino acid composition of type I collagen α1and α2 chains that form the type I collagen triple helix. The Pro-Hyp-Gly sequence (the corresponding amino acids are in bold) is the highest occurring sequence in the chains.1 Amino acids are organized by highest content in the α1(I) chain to lowest………………………...6 3. The stability effects of tertiary amides in comparison to POG and PPG sequences. Tertiary amides other than Nleu tend to have a destabilizing effect on the Tm of CMP triple-helices……………………………………………………………….14 4. Stereoelectronic effects contribute to the stability of the triple helix as shown by the use of fluorinated derivatives of proline. Tm values from Ref. 2……………16 5. Acp and Fmp were used to show that even though a residue may not have the appropriate ring puckering for the position it is in, the hydrogen bonding network can provide stability to the triple helix. Tm values from Ref. 45………………...18 viii LIST OF FIGURES Figure Page 1. Schematic of the biosynthesis of collagen. Genetic coding for collagen is transcribed to form a protocollagen strand. The protocollagen strand undergoes enzymatic modification to form a procollagen triple helix. Proteinases trim the N- and C-termini thus producing a tropocollagen triple helix which self-assembles into fibrils that cross-link to form fibers.2, 6 Reprinted with permission from Shoulder, MD, Raines, RT, Annual Review of Biochemistry, 2009, 78, 929-958 © 2009, Annual Reviews………………………………………………………….7 2. TEM and AFM images of collagen displaying D-periodicity. A) TEM of collagen fibrils from human Achilles tendon stained with gold-palladium. Scale bar = 500 nm. B) TEM of type I collagen from calk-skin stained with phosphotungstic acid. Scale bar = 100 nm. C) AFM image of corneal collagen fibrils. Scale bar = 100 nm. D) AFM image of scleral collagen fibrils. Scale bar = 100nm. A, B) Adapted with permission from Electron Microscopy Reviews, 3/1, Chapman, JA, et al., The collagen fibril – a model system for studing the staining and fixtion of a protein, 143-182 © 1990 with permission from Elsevier. C, D) Adapted with permission from Japanese Journal of Ophthalmology, 46/5, Yamamto, S, Hitomi, J, et al., Observation of human corneal and scleral collagen fibrils by atomic force microscopy, 496-501 © 2002 with permission from Elsevier…………………….8 3. A scheme of the helical twist and unit height parameters. In a 72 helical twist, there are 21 amino acids that constitute two turns of the helix and a unit height of 20 Å. Reprinted with permission from Biopolymers, 84/4, Okuyama, K, Wu, G, et al., Helical twists of collagen model peptides, 421-432 © 2006 John Wiley and Sons………………………………………………………………………………11 4. Cϒ-endo and Cϒ-exo ring puckering of the pyrrolidine ring residues. Proline or proline derivatives with Cϒ-endo ring puckering in the X position and Cϒ-exo ring puckering in the Y position provide stability to the collagen triple helix through stereoelectronic effects. These stereoelectronic effects orient the main chain dihedral angles of the collagen chain to form a triple helix…………….………..15 ix LIST OF FIGURES (CONT.) Figure Page 5. A schematic diagram of a one-residue stagger and interchain hydrogen bonding between three strands (dashed line) that enables triple helix formation in procollagen and (POG)n CMPs…………………………………………………..17 6. CMP design with a Gly→Ala substitution.49 This CMP mimics the defective collagen found in patients suffering from OI…………………………………….20 7. TEM of (POG)10 and polyPOG. A) (POG)10 with branched extensive branching of 64 65 the fibers. B) (POG)10 aggregates of nanofibers. C) poly-POG fibers 65 displaying higher dispersion of fibers than (POG)10. A) Reprinted with permission from The Journal of Biological Chemistry, 281/44, Kar, K, Amin, P, et al., Self-association of collagen triple helic peptides into higher order structures, 33283-33290 © 2006 American Society for Biochemistry and Molecular Biology B, C) Adapted with permission from Biopolymers, 79/3, Kishimoto, T, Morihara, Y, et al., Synthesis of poly (Pro–Hyp–Gly)n by direct polycondensation of (Pro–Hyp–Gly)n, where n= 1, 5, and 10, and stability of the triple‐helical structure, 163-172 © 2005 John Wiley and Sons……………….…25 8. TEM of nanofibers from a CMP with the sequence (GPP)3GPR-GEKGER- 72 GPR(GPP)3-GPCCG at different scales. A, B) Scale bar = 0.5 μm and C, D) Scale bar = 1μm. Reprinted with permission from Biomacromolecules, 32/7, Krishna, O. D., and Kiick, K. L., Integrin-mediated adhesion and proliferation of human MSCs elicited by a hydroxyproline-lacking, collagen-like peptide, 6412- 6424 © 2009 American Chemical Society........................................................…26 9. A) Schematic of PPG CMP F877. The underlined G at position 901 was substituted with Ser in the G901S CMP. B) TEM of F877 nanofibers. Scale bar = 40 nm. C) TEM of G901S fibers. Scale bar = 40nm. Adapted with permission from Angewandte Chemie International Edition, 46/18, Bai, H, Xu, K, et al., Fabrication of Au nanowires of uniform length and diameter using a monodisperse and rigid biomolecular template: collagen‐like triple helix, 3383- 3386 © 2007 John Wiley and Sons…………………………...………………….27 x LIST OF FIGURES (CONT.) Figure Page 10. A) TEM of the cubes assembled by streptavidin-labelled gold nanoparticles and a biotinylated F877 CMP. Scale bar = 1μm. B) TEM of the hexagonal structures that assembled when the diameter of the gold nanoparticles was raised to 30 nm. Scale bar = 1μm. Adapted with permission from Angewandte Chemie International Edition, 49/45, Kaur, P, Maeda, Y, et al., Three‐dimensional directed self‐assembly of peptide nanowires into micrometer‐sized crystalline cubes with