Characterization of Influenza Hemagglutinin Fusion Peptide and Transmembrane Domain Membrane Insertion Open Access

Prokopik, Alexia (2017)

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This project aimed to characterize the H1N1 fusion peptide and transmembrane domain in a model membrane environment. Influenza hemagglutinin is a necessary mediator for the membrane fusion step of the viral replication cycle, but many aspects of this mechanism are still unresolved. Though previously thought to be a passive anchor of hemagglutinin, the transmembrane domain is now thought to have a dynamic interaction with the fusion peptide to induce a hemifusion state in the host and viral membranes, ultimately leading to a fusion pore. The work in this thesis sought to characterize, using fluorescence emission, equilibrium Fourier transform infrared, and circular dichroism, each of these peptides individually in a model membrane system to understand how they interact with vesicles, neglecting the rest of the protein. It was demonstrated that the fusion peptide inserts into the vesicles at the melting temperature of the membranes, where the gel to fluid phase transition occurs. As the temperature increased, the peptide exited the membrane, forming aggregates and unfolding from the alpha-helical structure it adopted in the membrane. As for the transmembrane domain, it is still in the early stages of being characterized. However, it was demonstrated that the transmembrane domain could associate with the model membrane system and folds from a beta-hairpin structure to the expected alpha-helical conformation as reported in the literature. In the future, quenching experiments with the transmembrane domain will reveal its orientation within the membrane, and eventually lead to the characterization and dynamic study of the fusion peptide and transmembrane domain together in a model membrane system.

Table of Contents

Chapter 1: An Introduction to Membrane Proteins and Viral Fusion 1

1.1 Introduction 2

1.1.1 Membrane Protein Folding and Insertion 2

1.1.2 Viral Infection and Membrane Fusion 3

1.1.3 Influenza Hemagglutinin Structure and Role in Membrane Fusion 4

1.1.4 Fusion Peptide 6

1.1.5 Transmembrane Domain 7

1.1.6 The Interaction Between the Fusion Peptide and Transmembrane Domain 8

1.2 Conclusions and Aims 10

1.3 References 11

Chapter 2: Characterization of Fusion Peptide Insertion into Vesicles 14

2.1 Introduction 15

2.2 Materials and Methods 15

2.2.1 Peptide Synthesis and Purification 15

2.2.2 Lipid Vesicle Preparation 16

2.2.3 Fluorescence Emission 16

2.2.4 Circular Dichroism 17

2.2.5 Equilibrium Fourier Transform Infrared 17

2.3 Results and Discussion 18

2.3.1 Fluorescence Emission 18

2.3.2 Circular Dichroism 19

2.3.3 Equilibrium Fourier Transform Infrared 21

2.4 References 23

Chapter 3: Transmembrane Domain Characterization Within a Vesicle 25

3.1 Introduction 26

3.2 Materials and Methods 26

3.2.1 Peptide Synthesis, Purification, and Labeling 26

3.2.2 Lipid Vesicle Preparation 26

3.2.3 Fluorescence Emission 27

3.2.4 Circular Dichroism 27

3.3 Results and Discussion 27

3.4 References 30

Chapter 4: Conclusions and Perspectives 31

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