Cryo-EM as a tool to understand cross-alpha and beta nanotubes peptide systems Restricted; Files Only

Abstract

Chemical self-assembly is a necessary tool in the creation of de novo systems that would rival the level of complexity observed in nature. Within the scope of peptides nanomaterials, rational design is a vital means to help push the current boundaries within the protein design space. Cryo-EM, on the other hand, has become the standard rather than the exception in helping the scientific community bridge our knowledge gap of the structural interactions that govern diverse assembly systems, both natural and synthetic. To this end, several research projects will be presented here with the goal of contributing to the establishment of a set of design rules that would help us rationalize the relationship between sequence and the resulting structure of peptides nanotubes in ways that are both predictable and reproducible. These projects include the structural analysis of cross-a helical nanotubes as a means to gain insight into the designability of peptides materials, the structural and mechanistic study of cross-b sheet nanotubes from an amphipathic oligopeptide as well as an overview of cryo-EM as it applies to helical polymers. In addition to electron microscopy in all its forms, we employed a wide spectrum of biophysical analyses to study higher-order structures. These projects contributed to widening the structural and mechanistic “toolbox” for self-assembling peptide materials while providing a better understanding of the key principles in peptide assembly. 

Table of Contents

Chapter 1: Introduction

1.1 - The importance of peptides self-assemblies

1.1.1 - Peptides self-assemblies in biological systems

1.1.2 - Peptides self-assemblies in non-biological systems

1.2 - Structural characterization of helical assemblies

1.3 - Self-assembly of helical biomolecules

1.3.1 - Native helical assemblies

1.3.2 - Cross-a peptides nanotubes

1.3.3 - Cross-b peptides nanotubes

1.4 - Aims and scope of the dissertation

1.5 - References`

Chapter 2 : Structural analysis of a family of cross-helical nanotubes

2.1 - Introduction

2.2 - Result and discussion

2.2.1 - Structural differences between Form I and Form II Filaments b, a

2.2.2 - Dependence of filaments structure and peptide length

2.2.3 - Effect of arginine position

2.2.4 - Native designability of helical interfaces

2.2.5 - Structural comparison with other filamentous assemblies

2.3 - Conclusion

2.4 - Material and methods

2.4.1 - Peptide Synthesis, Purification, and assembly

2.4.2 - Circular dichroism spectropolarimetry

2.4.3 - Negative Stain Transmission electron microscopy Analysis

2.4.4 - Cryo-electron microscopy

2.4.5 - Model Building

2.4.6 -Motif Analysis

2.4.7 -Computational design

2.5 -Supplementary Figures

2.6 - Supplementary Tables

2.7 - References

Chapter 3: Self-assembly of a b-sheet nanotube from an amphipathic oligopeptide

3.1 - Introduction

3.2 - Result

3.2.1 - Different morphology of KFE8 nanotubes under cryo-EM

3.2.2 -The doubled-wall nanotubes are made of both parallel and anti-parallel sheets.

3.2.3 - Different helical packing from the same 8-residue peptide

3.3 - Discussion

3.4 - Conclusion

3.5 - Material and methods

3.5.1 - Peptide synthesis and assembly

3.5.2 - Circular dichroism spectropolarimetry

3.5.3 - Negative Stain TEM Analysis

3.5.4 - Motif Analysis

3.5.5 - Cryo-electron microscopy imaging and analysis

3.5.6 - Additional refinement of 1-protofilament

3.5.7 - Model building

3.6 – Supplemental figures and table

3.7 - References

3.8 Supplemental References

Chapter 4: Cryo-EM of helical Polymers

4.1 - Introduction

4.2 - Soft Matter

4.3 - Polymorphism

4.4 - Symmetry Determination

4.5 - Chaos

4.6 - Conclusion

4.7 - References

Chapter 5: Insight into the mechanism of formation of a b-sheet nanotube from an amphipathic oligopeptide

5.1 - Introduction

5.2 - Results

5.2.1 - There are multiple different populations of assemblies over time

5.2.2 - Conformational rearrangement of the peptides

5.2.3 - Reversibility of the assembly process until equilibrium

5.3 - Discussions

5.4 - Conclusion

5.5 - Materials and methods

5.5.1 - Peptide synthesis

5.5.2 -Peptide assemblies

5.5.3 - Circular dichroism

5.5.4 - Transmission electron microscopy

5.5.5 - Atomic Force Microscopy

5.5.6 - Static Light Scattering

5.5.7 - Fourier Transform Infrared Spectroscopy

5.6 - References

Chapter 6: Conclusion and outlook

6.1 - Conclusion

6.2 - Outlook

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Molecular Chemistry, Polymer Chemistry, Biochemistry
Palavra-chave	Cryo-EM
Committee Chair / Thesis Advisor	Bill Wuest, Emory University Vincent Conticello, Emory University David Lynn, Emory University

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