Dynamics of Influenza Virus Hemagglutinin Fusion Peptide Membrane Insertion Open Access

Anthony, Casey (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/pn89d722m?locale=en
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Abstract

The process of viral fusion is essential for understanding the protein-protein and protein-membrane interactions of influenza virus hemagglutinin and other analogous viral systems. The fusion peptide (FP) and the transmembrane domain (TMD) of HA2, particularly their membrane insertion and interaction, drive endosomal fusion and pore formation during the influenza virus replication process. Despite extensive studies of the hemagglutinin protein and this process, many questions surrounding FP and TMD and their mechanisms of action during membrane fusion remain. Particularly, the membrane and protein dynamics that govern these processes must be resolved.

This thesis aims to elucidate the time-resolved dynamics of fusion peptide insertion and the TMD-FP interaction. In order to probe the specific time-resolved dynamics of the TMD-FP interactions, it was necessary to first study where and at what rate the FP inserts in the membrane through equilibrium fluorescence spectroscopy and laser-induced temperature-jump spectroscopy. The data suggests that the FP inserts deeply in the membrane to at least the 10-carbon on the 16-carbon DPPC lipid chain on the microsecond timescale. In addition, the FP follows vectorial insertion in the membrane, with the N-terminus preceding the rest of the FP domain.

The last portion of this thesis begins to examine the phenomenon of J-aggregation of NBD dye in DPPC membranes. The desegregation of J-aggregates formed from high NBD concentrations occurs in association with the gel to fluid phase transition of the membrane and can be observed through a change in fluorescence with time-resolved dynamic experiments. This is important in furthering the study of membrane dynamics.These results will ultimately facilitate the study of the fusion peptide and its interaction with the transmembrane domain to elucidate the role of the TMD-FP complex in the membrane fusion process.

Table of Contents

Table of Contents
Chapter 1: An Introduction to Viral Membrane Proteins and Functions ..................
1

1.1. Introduction ......................................................................................................2

1.1.1. Dynamics of Protein-Membrane Interactions .................................2

1.1.2. Influenza Virus Hemagglutinin as a Model for Viral Fusion

Mechanisms ....................................................................................3

1.1.3. Viral Entry ...................................................................................... 4

1.1.4. Hemagglutinin Structure and Role in Protein-Mediated Fusion of

Influenza Virus................................................................................ 5

1.1.5. The Fusion Peptide ......................................................................... 6

1.1.6. HA2 Fusion Peptide and Transmembrane Domain Interaction....... 8

1.2. Conclusions and Aim of Dissertation ............................................................10

1.3. References...................................................................................................... 11

Chapter 2: Estimating H1N1 Fusion Peptide Insertion Depth Using FRET ............14

2.1 Introduction .........................................................................................15

2.1.1 Fusion Peptide Insertion .................................................................. 15

2.1.2 Experimental Approach ....................................................................15

2.2 Materials and Methods ....................................................................................17

2.2.1 Peptide Synthesis and Purification................................................... 17

2.2.2 Circular Dichroism ...........................................................................18

2.2.3 Vesicle Preparation ..........................................................................18

2.2.4 Equilibrium FTIR .............................................................................19

2.2.5 Equilibrium Fluorescence Emission .................................................19

2.2.6 Dynamic Light Scattering to Measure Fusion Activity ................... 19

2.3 Results and Discussion ...............................................................................................20

2.3.1 Effects of Fluorescent Probe NBD-PC on DPPC Structure and Phase Transition .........................................................................................20

2.3.2 Characterization of Fusion Peptide Secondary Structure by Circular Dichroism .........................................................................................21

2.3.3 Effect of NBD-Labeled Vesicles on Fusion Peptide Insertion ........ 23

2.3.4 Effect of TAMRA Label on Membrane Insertion of the Fusion Peptide............................................................................................... 24

2.3.5 Fusion Activity of Wild Type and Labeled Fusion Peptide ............27

2.3.6 Estimation of Insertion Depth with FRET ....................................... 28

2.4 Conclusions..................................................................................................... 30

2.5 References....................................................................................................... 31

Chapter 3: Probing the Kinetics of Fusion Peptide Insertion Using Fluorescence Temperature-Jump...32

3.1 Introduction .....................................................................................................33

3.1.1 Fusion Peptide Insertion and TMD Interaction ...............................33

3.1.2 Experimental Approach ....................................................................33

3.2 Materials and Methods.................................................................................... 34

3.2.1 Vesicle Preparation ..........................................................................34

3.2.2 Equilibrium FTIR............................................................................. 34

3.2.3 Equilibrium Fluorescence Emission ................................................ 35

3.2.4 Temperature Jump Fluorescence Spectroscopy ...............................35

3.3 Results and Discussion ................................................................................... 36

3.3.1 Effects of Brominated Lipids on DPPC Structure and Phase

Transition ......................................................................................... 36

3.3.2 Equilibrium Fluorescence Spectroscopy ..........................................37

3.3.3 Laser-induced Temperature Jump Fluorescence Spectroscopy ....... 38

3.4 Conclusions .....................................................................................................40

3.5 References .......................................................................................................41

Chapter 4: Probing the Dynamics of the Gel to Fluid Phase Transition in Unilamellar DPPC Vesicles Through Aggregation of NBD........................................41

4.1 Introduction..................................................................................................... 42

4.2 Materials and Methods.................................................................................... 43

4.2.1 Vesicle Preparation ..........................................................................43

4.2.2 Equilibrium Fluorescence Emission ................................................43

4.2.3 Temperature Jump Fluorescence Spectroscopy............................... 43

4.3 Results and Discussion ...................................................................................44

4.3.1 Equilibrium Fluorescence Detection of J-Aggregation ....................44

4.3.2 Temperature Jump Fluorescence Spectroscopy of NBD J -Aggregation............................................................................................... 46

4.4 Conclusions..................................................................................................... 47

4.5 References .......................................................................................................47

Chapter 5: Conclusions and Perspectives .....................................................................49

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