Biophysics of Membrane Remodeling driven by Protein Crowding Público

Abstract

Generation and stabilization of membrane curvature by proteins is considered to be indispensable for cellular function such as signaling, fusion and transport. Given the crowded nature of the interface, peripheral and trans-membrane proteins have been observed to trigger cellular shape changes as a consequence of their sheer density. Despite its prevalence in biology, key mechanistic details underlying membrane bending driven by protein crowding remain elusive.

In this thesis, a quantitative biophysical investigation of membrane bending driven by protein crowding is presented. We developed a quantitative fluorescence-based assay involving energy transfer between donor-tagged lipids (NBD-PE) and acceptor-tagged lipids (Rhodamine-PE) incorporated into the membrane surface. This approach provided a spectroscopic handle on membrane area expansion, thus enabling ensemble measurements in the bulk solution phase, instead of a conventional imaging-based approach. Using this methodology, we quantitatively determine the critical protein-lipid ratios at which membrane bending was triggered. We find strong coupling between the intrinsic vesicle curvature and the phase behavior of the liposomes used, indicating that curvature stress imposed by changing the vesicle diameters can affect membrane deformation. While these equilibrium measurements provide a handle over several experimental variables, little information could be obtained with regards to presence of structural intermediates that precede membrane deformation.

To address this, we quantified the association kinetics of protein binding to a liposome surface using Stopped-flow fluorescence quenching. Results from our study indicates that protein binding to lipid surface does not follow a simple bi-molecular association behavior. Instead, protein binding occurs through multiple, interconvertible conformations with varying packing densities. Finally, we extend our kinetic investigation to measure the timescales of membrane deformation using stopped-flow fluorescence. We report a complex multi-phasic behavior, with the vesicles sampling at least two intermediates before reaching its final deformed state. Subsequent biophysical investigation reveals that protein binding causes membrane thinning, before resulting in deformation of the vesicle structure. Together with the equilibrium data, our results greatly enhance the fundamental understanding of membrane deformation driven by protein crowding.

Table of Contents

Chapter 1: Introduction to membrane remodeling by protein attachment….1

1.1          Lipid membrane morphology and its significance …………………………………………..2

1.1.1 Lipid membranes as biological envelopes………………………………………………..2

1.1.2 Cellular traffic and membrane curvature…………………………………………………3

1.1.3 Membrane curvature in pathophysiology………………………………………………..4

1.1.4 Physicochemical properties that govern membrane curvature and protein binding…….…5

1.2          Protein driven membrane remodeling- The classic mechanisms……………………….6

1.2.1 Cytoskeletal protein driven shape changes………………………………………………7

1.2.2 Modulation of lipid composition and asymmetry by protein association……8

1.2.3 Insertion of hydrophobic regions of amphipathic proteins……………………….8

1.2.4 Curvature sensing and scaffolding proteins……………………………………………9

1.3          Membrane bending driven by protein crowding……………………………………………10

1.3.1 Ubiquity of the crowded membrane-protein interface…………………………….10

1.3.2 Membrane curvature generation by protein crowding…………………………….11

1.3.3 Fluctuations in membrane bound protein structure and its role in curvature generation……………………13

1.3.4 Membrane curvature generation accomplished by protein unfolding-folding transitions.………………….14

1.3.5 Debate in the literature and the need for mechanistic investigation…………15

1.4          Aims and scope of this thesis……………………………………………………………………….17

1.5          References………………………………………………………………………………………………….19

Chapter 2: Curvature regulated phase separation in binary lipid mixtures, and its role in membrane bending ………24

2.1 Introduction……………………………………………………………………………………………………25

2.2 Results and Discussion……………………………………………………………………………………29

2.2.1 Design and optimization of donor-acceptor doping levels for the FRET assay……………………29

2.2.2 Spectroscopic observation of membrane area expansion………………………..31

2.2.3 Vesicle intrinsic curvature modulates critical deformation concentration..34

2.2.4 Small Angle X-Ray Scattering reveals that the bilayer thickness and lamellarity is unaffected by vesicle curvature……………36

2.2.5 Curvature regulated phase-behavior in binary mixtures observed by DPH quenching………………………………………………….39

2.2.6 Coupling between membrane bending driven by protein binding and the vesicle phase structure……………………………………45

2.3 Conclusion…………………………………………………………………………………………………….46

2.4 Experimental Section……………………………………………………………………………………..47

            2.4.1 Materials………………………..…….…………………………………………………………..47

            2.4.2 Protein expression and purification.…….…….…….…….…….…….…….………..47

            2.4.3 Large Unilamellar vesicles preparation……….…….………...…….……...…….….48

2.4.4 Dynamic Light Scattering measurements.…….……….…….………………………49

2.4.5 Equilibrium fluorescence measurements for CDC determination……………49

2.4.6 Fluorescence lifetime measurements.…….………..…….…………………………….50

2.4.7 Isothermal Titration Calorimetry……………………...…….…………………………..50

2.4.8 Observation of domain formation by DPH quenching……………………………51

2.4.9 Approximation of domain radius…….…….…………..…….…………..…….……….52

2.4.10 Small Angle X-Ray Scattering…...…….…………..…….………...…….…………….52

2.5 References………...…….……………..…….……………..…….………………...…….…………………53

Appendix 2: Curvature regulated phase separation in binary lipid mixtures, and its role in membrane bending…….…………59

Chapter 3: Kinetics of histidine tagged protein association to nickel-decorated liposome surfaces…………………………63

3.1 Introduction.……...…….……………..…….……………..…….………………...…….………………..64

3.2 Results and Discussion.……...…….……………..…….……………..…….………………………….68

           3.2.1 Equilibrium characterization of liposome-SfGFP interaction………………….68

           3.2.2 Stopped-Flow fluorescence reveals a complex, multi-phasic binding

           behavior……..…….………………..…….………………..…….………………..…….……………….70

           3.2.3 The role of chelator lipid density on association kinetics………………………..73

           3.2.4 Understanding the effects of lipid lateral mobility on association kinetics.76

           3.2.5 Protein surface charge does not significantly modify the association kinetics at high protein densities.……..80

3.2.6 Protein binding to the liposome surface can occur via multiple conformations…..…….………………..…….…………..81

3.3 Conclusions…..…….………………..…….…………..…….………………..…….………………………88

3.4 Experimental Section…..…….………………..…….…………..…….………………..…….………..89

           3.4.1 Materials…..…….………………..…….…………..…….………………..…….………………89

           3.4.2 Large Unilamellar vesicles preparation………..…….………………..…….………..89

           3.4.3 Dynamic light scattering measurements…..…….………………..…….……………90

           3.4.4 Steady state fluorescence measurements…..…….………………..…….……………90

           3.4.5 Stopped flow fluorescence measurements.…..…….………………..…….…………90

           3.4.6 Theoretical estimation of protein footprint on membrane surfaces…………91

           3.4.7 Protein expression and purification…..…….………………..…….…………………..92

           3.4.8 Isothermal Titration Calorimetry…..…….………………..…….………………………93

           3.4.9 Analysis of steady state fluorescence data …..…….………………..…….………….94

           3.4.10 Fluorescence recovery after photobleaching. …..…….………………..…….……95

           3.4.11 Preparation of Cu and Co containing liposomes……………………………………95

           3.4.12 Analysis of stopped flow transients…..…….………………..…….………………....96

           3.4.13 Anisotropy correction…..…….………………..…….……………………………………..96

3.5 References…..…….………………..…….……………………………….………………..…….………....97

Appendix 3: Kinetics of histidine tagged protein association to nickel decorated liposome surfaces.……………104

Chapter 4: Membrane thinning precedes membrane bending by protein crowding……………….……………113 

4.1 Introduction.……...…….……………..…….……………..…….………………...…….………………..114

4.2 Results and Discussion.……...…….……………..…….……………..…….…………………………116

           4.2.1 Equilibrium characterization of liposome deformation driven by protein crowding…...…116

4.2.2 Stopped flow transients reveal an unresolved intermediate that precedes membrane deformation……..…….…118

4.2.3 Synthesis and characterization of Asymmetric vesicles………………………..121

4.2.4 Temporal separation of membrane thinning and membrane bending events by stopped flow fluorescence……..124

4.2.5 Characterization of change in membrane thickness by Small-Angle X-Ray Scattering.……..…….……………..……127

4.3 Conclusions……..…….……………..…………..…….…………………………………………………..130

4.4 Experimental Section…………………………………………………………………………………….131

           4.4.1 Materials………………………………………………………………………………………….131

           4.4.2 Large Unilamellar vesicles preparation……………………………………………….132

           4.4.3 Dynamic light scattering measurements……………………………………………..132

           4.4.4 Equilibrium fluorescence measurements…………………………………………….133

           4.4.5 Stopped flow fluorescence measurements……………………………………………133

           4.4.6 Asymmetric vesicle preparation…………………………………………………………134

           4.4.7 Small Angle X-Ray Scattering measurements………………………………………135

4.5 References…………………………………………………………………………………………………….135

Appendix 4: Membrane thinning precedes membrane bending by protein crowding…139

Chapter 5: Conclusion and Perspectives.………………………………………...….141

5.1 Summary……………………………………………………………………………………………………..142

5.1.1 Exploring the physicochemical parameters that regulates membrane bending driven by protein crowding………………….143

5.1.2 Kinetic intermediates that precede membrane bending………….…………..143

5.2 Future Outlook……………………………………………………………………………………………145

           5.2.1 Kinetics of vesicle fission and fusion.………………………………………………..145

           5.2.2 Rational design of modular bio-conjugates……..………………………………..145

5.3 References.…………………………………………………………………………………………………146

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biophysics, General Chemistry, Physical
Palabra Clave	Lipid-protein interaction Membrane bending Membrane remodeling Membrane curvature Membrane thinning Protein crowding
Committee Chair / Thesis Advisor	R. Brian Dyer, Emory University
Committee Members	James T. Kindt, Emory University Khalid Salaita, Emory University

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