Engineering Self-Assembling Peptide Systems Público

Modlin, Charles (Fall 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/47429b11f?locale=pt-BR
Published

Abstract

Chemical self-assembly is an invaluable tool used in the bottom-up creation of de novo systems that otherwise would be impossible to construct in a top-down approach given the size regime in which many biomaterials exist. Currently, the ability to engineer self-assembling systems that rival the level of complexity observe in nature is highly limited. However, rational design enables researchers to push the current limits of complexity within de novo self-assembling systems, specifically within the scope of self-assembling peptide biomaterials. Often, this involves using what is observed in nature as a foundation for further design. To this end, numerous research projects will be presented, which hinge upon the rational design and alteration of existing systems to produce new and novel systems. These projects include the use of an arginine staple motif as a means of engineering nanotubular structures with variable diameters and structures, the use of computational design to create entirely new sequences which self-assemble into solenoidal nanotubes, and the addition of DNA to peptide systems as a means of increasing the complexity of self-assembling systems beyond what is possible for single-species assembly. To verify the hypotheses regarding these systems, a wide spectrum of biophysical analyses was used. The resultsof these projects help widen the structural ‘tool box’ for self-assembling peptide materials, and help provide better understanding of the key principles in peptide self-assembly.

Table of Contents

Chapter 1. Introduction to Self-Assembly and Helical Supramolecular Assemblies

1.1 The role and importance of self-assembly in biological systems 1

1.2 Self-assembling helical biomolecules 3

1.2.1 Native helical biomolecules 3 1.2.2 Non-native helical assemblies from synthesized peptides 9

1.3 Helical assemblies beyond peptides 15

1.4 Conclusion 20

1.5 References 22

Chapter 2. Development and Characterization of Forms I and II

2.1 Introduction 29 2.2 Sequence Design 30 2.3 Results and Discussion 32

2.3.1 Circular Dichroism 32 2.3.2 Transmission Electron Microscopy 33 2.3.3 Small- and Wide-Angle X-Ray Scattering 34 2.3.4 Cryo-Electron Microscopy and Helical Reconstruction 37 2.3.5 Power Spectra and Reference-Free Class Averages 42

2.4 Conclusion 44 2.5 Methods 45

2.5.1 Peptide Synthesis 45 2.5.2 EM and Image Analysis 46 2.5.3 Atomic Models 49 2.5.4 Synchotron SAXS/WAXS measurements 50 2.5.5 Accession numbers 51

2.6 References 52

Chapter 3. Expanding the Form Peptide Series: Engineering Self-Assembling HelicalPeptide Nanotubes Using An ‘Arginine Staple’ Structural Motif

3.1 Form III Introduction 56

3.2 Form III Sequence Design 62

3.3 Form III Results and Discussion 64

3.3.1 Peptide Self-Assembly 64 3.3.2 Circular Dichroism and Flow Linear Dichroism 65 3.3.3 Transmission Electron Microscopy 69 3.3.4 Scanning Transmission Electron Microscopy 71 3.3.5 Power Spectra and Reference-Free Averages 73 3.3.6 Small- and Wide-Angle X-Ray Scattering 75

3.4 Form III Conclusion 79 3.5 Form IV: Testing the Upper Size Limit of the Arginine Staple in Sequence Space 81

3.5.1 Introduction and Sequence Design 81 3.5.2 Results and Discussion 82

3.5.2.1 Circular Dichroism 82 3.5.2.2 Transmission Electron Microscopy 83

3.5.3 Conclusion 85

3.6 Form 0: Testing the Arginine Staple in Shorter Helices 86

3.6.1 Introduction and Sequence Design 86 3.6.2 Results and Discussion 88

3.6.2.1 Circular Dichroism 88

3.6.2.2 Transmission Electron Microscopy 89

3.6.2.3 Small and Wide-Angle X-Ray Scattering 90

3.6.2.4 Conclusion 93

3.7 Methods 93

3.7.1 Materials 93 3.7.2 Peptide Self-Assembly 93 3.7.3 Circular Dichroism 101 3.7.4 Flow Linear Dichroism 101 3.7.5 Transmission Electron Microscopy 102 3.7.6 Scanning Transmission Electron Microscopy 103 3.7.7 Small- and Wide-Angle X-Ray Scattering 103

3.8 References 105

Chapter 4. Form IA: A Concentration Dependent Switch Between Tube and Crystal

4.1 Introduction 111

4.2 Results and Discussion 113

4.2.1 Peptide Self-Assembly 113 4.2.2 Circular Dichroism and Flow Linear Dichroism, Low Concentration 113 4.2.3 Circular Dichroism, High Concentration 118 4.2.4 Transmission Electron Microscopy, Low Concentration 120 4.2.5 Transmission Electron Microscopy, High Concentration 121 4.2.6 Scanning Transmission Electron Microscopy 127 4.2.7 Small -Angle X-Ray Scattering 129

4.3 Conclusion 130

4.4 Methods 131

4.4.1 Peptide Synthesis 131 4.4.2 Peptide Self-Assembly 133 4.4.3 Circular Dichroism 135 4.4.4 Transmission Electron Microscopy 135 4.4.5 Scanning Transmission Electron Microscopy 136 4.4.6 Small- and Wide-Angle X-Ray Scattering 137

4.5 References 139

Chapter 5. Improving Designability and Predictability of Self-Assembling Peptide Systems: Computationally Designed Solenoidal Nanotubes Based on an Alpha-Loop-Beta Structural Motif

5.1 Introduction 141

5.2 Results and Discussion 144

5.2.1 Sequence and Assembly Design 144 5.2.2 Circular Dichroism 153 5.2.3 Transmission Electron Microscopy 155 5.2.4 Scanning Transmission Electron Microscopy 159 5.2.5 Small-Angle X-Ray Scattering 161 5.2.6 Cross-Sectional Pair Distance Distribution Function 166

5.3 Ongoing Work 168

5.4 Conclusion 171

5.5 Methods 173

5.5.1 Materials 173 5.5.2 Sequence Design 173 5.5.3 Peptide Self-Assembly 178 5.5.4 Circular Dichroism 183 5.5.5 Transmission Electron Microscopy 183 5.5.6 Scanning Transmission Electron Microscopy 184 5.5.7 Small- and Wide-Angle X-Ray Scattering 185

5.6 References 186

Chapter 6. Increasing Structural Complexity with Self-Assembling Systems of Multiple

Species: Three-Dimensional Peptide-DNA Hybrid Assemblies

6.1 Introduction 194

6.2 Results and Discussion 198 6.2.1 CP+-TL Hybrid: A first attempt at controlled co-assembly of peptide and nucleic material 198

6.2.1.1 Circular Dichroism of CP+ 199 6.2.1.2 Transmission Electron Microscopy of CP+ and CP+ - TL 201

6.2.2 CP++- TL and sCP++- TL: A new approach for creating DNA-peptide hybrid nanowires 204

6.2.2.1 Circular Dichroism of CP++ and sCP++ 204 6.2.2.2 Transmission Electron Microscopy of CP++- TL 206

6.2.2.3 Transmission Electron Microscopy of Varying Ratios of CP++ and TL 208

6.2.2.4 Small- and Wide-Angle X-Ray Scattering of CP++ - TL 212

6.2.2.5 Transmission Electron Microscopy: sCP++-TL 214 6.2.3 A DNA design for ribbon formation: the DNA-brick 215

6.2.3.1 Transmission Electron Microscopy of CP++-DNAbrick 216 6.3 Conclusion 219

6.4 Methods 219

6.4.1 Materials 219 6.4.2 Peptide Synthesis 220 6.4.3 TL Nanosheet Preparation 220 6.4.4 2Hx8H and 4Hx8H Preparation 221 6.4.5 Co-assembly of Peptide and DNA 221 6.4.6 Circular Dichroism 221 6.4.7 Transmission Electron Microscopy 222 6.4.8 Small- and Wide-Angle X-Ray Scattering 222 6.4.9 DNA Sequences for TL and Brick Structures 223

6.5 References 224

Chapter 7. Conclusion and Outlook

7.1 Conclusion 227

7.2 Outlook 228

7.3 References 229

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Degree
Submission
Language
  • English
Research Field
Palavra-chave
Committee Chair / Thesis Advisor
Committee Members
Última modificação

Primary PDF

Supplemental Files