Investigating Influenza Hemagglutinin Fusion Peptide Insertion into Model Membrane Liposomes with Fluorescence and FTIR Spectroscopy Public
Anderson, Catharine (Spring 2022)
Published
Abstract
The highly conserved first 23 residues of influenza hemagglutinin (HA), known as the fusion peptide (FP), are critical for fusion of the viral and endosomal membranes during viral infection. Even conservative substitutions in the FP have been shown to significantly alter the peptide’s conformational ensemble and abolish fusogenicity. Equilibrium fluorescence and FTIR spectroscopy are used here to study the effects of different point mutations on fusion peptide insertion into vesicles. Insertion is initiated by heating DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) lipid vesicles above the gel-to-liquid phase transition temperature of DPPC. In addition to the WT peptide, this study examines the variants G1V, W14A, G8A, and G16A. Each of these point mutations is expected to disrupt the tight helical hairpin structure of the WT FP. The G1V and W14A variants, which are expected to sample wide-angle open conformations, appear to insert more shallowly than WT or allow more water leakage into the membrane. The G8A and G16A variants, which are expected to sample acute-angle open conformations, appear to partially insert at lower temperatures than WT and disorder the membrane lipid tails to a greater extent. Fusion peptide insertion appears to be irreversible, most likely due to the disordering of the peptides and membrane environment at higher temperatures. With the equilibrium signals characterized, time- resolved temperature-jump fluorescence and FTIR spectroscopy could be used to visualize the timescale of insertion for each fusion peptide variant. Resolving the temporal and spatial details of fusion peptide insertion could eventually enable the development of a universal influenza treatment.
Table of Contents
Chapter 1: Introduction to Hemagglutinin-Mediated Viral Membrane Fusion..........1
1.1 Introduction..........2
1.1.1 Influenza Hemagglutinin Structure and Role in Membrane Fusion..........2
1.1.2 Wild Type Fusion Peptide Structure..........5
1.1.3 Fusion Peptide Variants Structure..........5
1.2 Conclusions and aims..........8
1.3 References..........8
Chapter 2: Characterization of Fusion Peptide Insertion into Model Membrane Liposomes with Fluorescence Spectroscopy ..........2
2.1 Introduction..........13
2.2 Materials and Methods..........14
2.2.1 Model Membrane Liposome Preparation..........14
2.2.2 Fusion Peptide Preparation..........14
2.2.3 Fluorescence Emission..........15
2.3 Results and Discussion..........16
2.3.1 FP Insertion Melt Experiments..........16
2.3.2 FP Insertion Reversibility Experiments..........21
2.4 References..........25
Chapter 3: Characterization of Fusion Peptide Insertion into Model Membrane Liposomes with FTIR Spectroscopy..........26
3.1 Introduction..........27
3.2 Materials and Methods..........27
3.2.1 Model Membrane Liposome Preparation..........27
3.2.2 Fusion Peptide Preparation..........28
3.2.3 FTIR..........28
3.3 Results and Discussion..........29
3.3.1 FTIR Peptide Amide Region..........29
3.3.2 FTIR Lipid Tail CH Stretch Region..........33
3.3.3 FTIR Lipid Headgroup Carbonyl Stretch Region..........35
3.3.4 FTIR Reversibility Experiment..........38
3.4 References..........39
Chapter 4: Conclusions and Perspectives..........41
4.1 Conclusions and Perspectives..........42
4.2 References..........44
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