Engineering Self-Assembling Peptide Systems with Antimicrobial Potential Público

Abstract

The alarming rise in antibiotic resistance over the past few decades has led to an urgent global health concern, creating a constant need for new therapeutic strategies. Engineering natural peptides found in organisms offers a promising alternative to existing therapeutics, which are susceptible to antimicrobial resistance. Antimicrobial peptides (AMPs) found in the innate immune system of vertebrates have been key in allowing the organisms to evolve in the way they did. AMPs possess substantial antimicrobial properties that treat various pathogens. The chances of pathogens developing resistance towards these peptides are less likely since peptides act upon a wide range of cellular components, whereas conventional therapeutics have one specific target. AMPs mainly form charged, hydrophobic and amphipathic structures which contribute to their bioactivity. However, stability, activity and cell selectivity need to be improved in order to make such peptides more attractive for therapeutic applications. Here, extensive efforts are being made to determine optimized assembly conditions for AMPs. These hierarchical structures possess a higher density of bioactive characteristics than their monomeric counterparts. Currently, the ability to predict the final assembly structure, from the primary peptide sequence, remains a significant challenge. We aim to develop design principles specifically directed for assembling AMPs. Understanding the correlation between sequence amphiphilicity, self-assembly behavior, and antimicrobial activity, will enable us to engineer more potent AMPs. Furthermore, these AMP assemblies, will allow us to study the relationship of antimicrobial activity and assembly structure. We propose that the self-assembly of engineered AMPs will result in improved antimicrobial activity and elucidate new mechanisms for combating pathogens. Natural peptides that have been previously shown to have antimicrobial and self-assembly properties (PSMα3 and CL1) are rationally designed and assembled into supramolecular structures to gain further insights into their structure-function relationship. We find that these peptides form different structures such as nanotubes, filaments and twisted ribbons. Complete structural characterization of the AMP assemblies and assessment of their antibacterial activities is executed. 

Table of Contents

Chapter 1. Introduction

1.1 Antimicrobial resistance and the urgent need for alternative therapies............................1

1.1.1 Limitations of current antibiotics for the treatment of bacterial infections...........2

1.2 Introduction to self-assembly of biological molecules................................................3

1.3 Antimicrobial peptides....................................................................................8

1.3.1 Antimicrobial peptides interactions with cell membranes.................................9

1.3.2 The limitations of antimicrobial peptides and how self-assembled structures can overcome them............11

1.4 Conclusion................................................................................................15

1.5 References................................................................................................16

Chapter 2. Development and Characterization of CL1 Peptides

2.1 Introduction...............................................................................................19

2.2 Sequence Design.........................................................................................20

2.3 Results and Discussion

2.3.1 Transmission Electron Microscopy.........................................................21

2.3.2 Circular Dichroism...........................................................................23

2.3.3 Scanning Transmission Electron Microscopy.............................................23

2.3.4 Small-and Wide-Angle X-Ray Scattering.................................................24

2.3.5 Proposed Atomic Models of CL1_2A Nanotubes.........................................27

2.4 Conclusion................................................................................................31

2.5 Methods

2.5.1 Materials.......................................................................................32

2.5.2 Peptide Synthesis..............................................................................32

2.5.3 Preparation of assemblies....................................................................32

2.5.4 Electron microscopy..........................................................................33

2.5.5 Circular Dichroism...........................................................................33

2.5.6 Synchrotron SAXS measurements.........................................................34

2.6 References................................................................................................35

Chapter 3. Development and Characterization of CL1

3.1 Introduction...............................................................................................36

3.2 Sequence Design.........................................................................................37

3.3 Results and Discussion

3.3.1 Transmission Electron Microscopy.........................................................37

3.3.2 Circular Dichroism...........................................................................39

3.3.3 Scanning Transmission Electron Microscopy.............................................41

3.3.4 Small-and Wide-Angle X-Ray Scattering.................................................41

3.3.5 Atomic Force Microscopy...................................................................43

3.3.6 MALDI-TOF Mass Spectrum...............................................................44

3.3.7 Proposed Atomic Model of PSMα3_4R Nanotubes......................................48

3.4 Conclusion................................................................................................50

3.5 Methods

3.5.1 Materials.......................................................................................51

3.5.2 Peptide synthesis..............................................................................51

3.5.3 Preparation of Assemblies....................................................................51

3.5.4 Electron Microscopy.........................................................................51

3.5.5 Circular Dichroism............................................................................52

3.5.6 Synchrotron SAXS measurements..........................................................52

3.5.7 Atomic Force Microscopy...................................................................53

3.5.8 MALDI-TOF Mass Spectrometry..........................................................53

3.6 References................................................................................................54 

About this Master's Thesis

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School	Laney Graduate School
Department	Chemistry
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Chemistry, Biochemistry Biology, Microbiology
Palabra Clave	antimicrobial Self-assembly
Committee Chair / Thesis Advisor	Conticello, Vincent, Emory University
Committee Members	Ke, Yonggang , Emory University Heemstra, Jen, Emory University

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