Investigating Fundamental Mechanisms of Peptide Insertion into Membranes Open Access

Schuler, Erin Elizabeth (2015)

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Thirty percent of the human genome is committed to production of membrane proteins. The importance of membrane proteins transcends all living organisms as these macromolecules serve as the gatekeepers to many processes that maintain homeostasis and cellular communication. Essential to adopting proper structure and function is the ability for membrane proteins to insert into target membranes. Despite the significance of this process, many questions concerning the fundamental mechanism of how insertion is achieved remain. For example, how do dynamic protein structures facilitate insertion? What are the energetics that govern this process? What role does the membrane play? Perhaps an even greater deficit in the field is the ability to couple membrane dynamics to those of proteins. Both membranes and proteins represent complex classes of macromolecules. In addition, the dynamics that govern these structures challenge the temporal resolution of many available techniques often used to study these systems. Thus, the nature of protein-membrane interactions necessitates development of time-resolved methods capable of capturing such dynamic processes. This dissertation aims to examine the mechanisms that drive spontaneous insertion of peptides into membranes, coupling membrane dynamics to those of the inserting peptides. In brief, the first half of this dissertation focuses on characterizing the dynamics of the fluid phase transition of model membranes and then utilizes the physical properties of membrane phases to examine spontaneous insertion of a small antimicrobial peptide, Mastoparan X (MPX). To accomplish this, the gel to fluid phase transformation was characterized in model d62-DPPC unilamellar vesicles by temperature-jump infrared spectroscopy. These studies revealed that d62-DPPC vesicles achieve the fluid phase state in only hundreds of nanoseconds by propagation of disorder from the edges of the membrane facets. Utilizing the ability to rapidly trigger the fluid phase state of the d62-DPPC membranes, the insertion rate of MPX was determined. These results indicate that insertion occurs within a few hundred microseconds, and importantly, determine the thermodynamic parameters that govern MPX insertion into a model membrane thereby establishing an exciting new methodology to probe spontaneous insertion of proteins into membranes. The second half of this dissertation examines peptide insertion as it relates to the mechanism of influenza A hemagglutinin (HA) protein, and its role in viral membrane fusion. It is hypothesized that early steps in HA-mediated membrane fusion are driven by folding of the coiled-coil motif Loop-40 (L40) in response to acidification of the endosome upon viral uptake. Ultimately, this process promotes extension of the HA fusion peptide to the surface of the host endosome, priming insertion of HA. In this dissertation, the folding mechanism of HA L40 peptide was studied by temperature-jump infrared and fluorescence spectroscopy. These results reveal a complex folding pathway, where three possible intermediates are accessed between the folded and unfolded state. Additionally, formation of the coiled-coil core is rapidly achieved, taking only ~10-4 s to fold. Lastly, an infrared fusion assay was successfully developed using combinations of isotope labeled and non-isotope labeled lipids to establish a platform for simultaneous monitoring of HA driven insertion and membrane fusion. Together, these studies add to the fundamental understanding of dynamic protein-membrane interactions, focused specifically on the underlying mechanisms that govern spontaneous insertion of peptides into membranes.

Table of Contents

Chapter 1: A General Introduction to Dynamic Protein-Membrane Interactions 1

1.1 Introduction 2

1.1.1 Historical Perspective

1.2 Membrane Theory 3

1.2.1 The Fluid Mosaic Model 3

1.2.2 The Mattress Model 3

1.2.3 Modern Perspective on Membrane Theory: Macromolecular Crowding

and Lipid Order 4

1.3 Dynamic interactions between membranes and proteins enhanced by lipid order 4

1.3.1 Hydrophobic Matching 4

1.3.2 Membrane Protein Folding 5

1.3.3 Insertion of Proteins into Membranes 6

1.4 Biomedical Relevance of Protein insertion into Membranes: Viral Pathogenicity, Antimicrobial Peptides and Therapeutics 6

1.5 Aim and Scope of Dissertation 7

1.6 References 8

Chapter 2: Dynamics of the Gel to Fluid Phase Transformation in Unilamellar DPPC Vesicles 12

2.1 Introduction 13

2.2 Materials and Methods 15

2.2.1 Preparation of d62-DPPC LUVs 16

2.2.2 Equilibrium FTIR 17

2.2.3 Temperature-Jump Infrared Spectroscopy 17

2.2.4 Simulation Methods 18

2.3 Results and Discussion 19

2.3.1 Equilibrium FTIR 19

2.3.2 Temperature-jump Infrared Spectroscopy 22

2.3.3 Simulations 27

2.4 Conclusions 32

2.5 References 33

Appendix 2: Dynamics of the gel to liquid-crystal phase transformation in lipid bilayer vesicles 36

A2. 1 Additional Figures 36

A2.2 Additional Information on Fitting Parameters 37

A2.2.1 Relationship Between Fitting Parameters in Equation 2 37

2.2.1 Derivation of Single Exponential Form for Vesicle Melting 37

Chapter 3: Insertion of Mastoparan X into Model d62-DPPC Lipid Vesicles Triggered by Rapid Thermal Initiation of the Membrane Phase Transition 39

3.1 Introduction 40

3.1.1 Selection of a Model Peptide 41

3.1.2 Experimental Approach 43

3.2 Materials and Methods 44

3.2.1 Peptide Synthesis and Purification 44

3.2.2 Lipid Vesicle Preparation 44

3.2.3 Circular Dichroism (CD) 45

3.2.4 Equilibrium Fluorescence 45

3.2.5 Equlibrium Fourier Transform Infrared (FTIR) spectroscopy 45

3.2.6 Temperature-Jump Fluorescence Spectroscopy 46

3.2.7 Time-Resolved Temperature Jump Infrared Spectroscopy 46

3.3 Results and Discussion 47

3.3.1 Characterization of MPX Folding by Circular Dichrosim (CD) 47

3.3.2 Membrane dependent MPX Fluorescence Emission 49

3.3.3 Probing Peptide Insertion by Equilibrium FTIR 50

3.3.4. Time-resolved Temperature Fluorescence Spectroscopy 53

3.3.5 Insertion Probed by Time-Resolved T-Jump Infrared Spectroscopy 54

3.4 Conclusions 58

3.5. References 60

Appendix 3: Insertion of Mastoparan X into Model d62-DPPC Lipid Vesicles Triggered by Rapid Thermal Initiation of the Membrane Phase Transition 63

A3.1 Sulforhodamine B Leakage Assay 63

Chapter 4: Characterization of the Folding Dynamics of Hemagglutinin

HA2 L40 peptide 66

4.1 Introduction 67

4.1.1 Hemagglutinin Structure and Function 67

4.1.2 Models of HA Folding 68

4.1.3 Loop-40 Coiled-coil 68

4.2 Materials and Methods 71

4.2.1 Peptide Synthesis and Purification 71

4.2.2. Circular Dichroism 71

4.2.3 Analytical Ultracentrifugation 72

4.2.4 Fluorescence Emission 72

4. 2.5 Equilibrium FTIR 72

4.2.6 Temperature-Jump Infrared Spectroscopy 73

4.2.7 Temperature Jump Fluorescence Spectroscopy 73

4.3 Results and Discussion 74

4.3.1 Characterization of Secondary Structure by Circular Dichroism 74

4.3.2 Analytical Ultracentrifugation 76

4.3.3 pH Dependent Fluorescence Emission Spectra 79

4.3.4 Equilibrium FTIR 81

4.3.5 Kinetic Measurements by Temperature-Jump

Infrared/Fluorescence Spectroscopy 82

4.4 Conclusions 88

4.5 References 89

Appendix 4: Characterization of the Folding Dynamics of Hemagglutinin HA2 L40 peptide 94

Chapter 5: Development of an FTIR Assay for Monitoring HA Fusion

Peptide Mediated Membrane Fusion 96

5.1 Introduction 97

5.1.1 Membrane Fusion 97

5.1.2 Viral Membrane Fusion 98

5.1.3 Current Approaches to Studying Membrane Fusion 99

5.2 Materials and Methods 100

5.2.1 Peptide Synthesis and Purification 100

5.2.2. Circular Dichroism 100

5.2.3 Fluorescence Emission 101

5. 2.4 Equilibrium FTIR 101

5.3 Results and Discussion 101

5.3.1 Confirmation of Secondary Structure by Circular Dichroism 101

5.3.2 Confirmation of Insertion by Fluorescence Emission Spectra 102

5.3.3 Infrared Fusion Assay 103

5.4 Conclusions and Future Directions 108

5.5 References 108

Chapter 6: Conclusions and Perspectives 112

6.1 Summary 113

6.2 Future Outlook 114

6.3 Other Contributions 115

6.4 References 116

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