A Spin Probe EPR Perspective on α-Synuclein Dynamics and Structure Under Controlled Confinement in Monomeric, Oligomeric, and Fibrillar Forms Restricted; Files Only

Abstract

“Structure determines function,” but what about intrinsically disordered proteins (IDPs), that lack a defined structure? α-Synuclein (αS), a 140 amino acid protein, which aids in neurotransmitter release, and whose dysfunction is associated with Parkinson’s disease (PD) in brain neurons. It is intrinsically disordered in its monomeric form but adopts multimeric functional forms and dysfunctional cytotoxic oligomer and structurally-related fibrillar forms, with an increasing degree of β-sheet core structure. Core structure is formed by the non-amyloid component (NAC; residues 61-95), which is flanked by intrinsically disordered regions (IDRs) in the N-terminal domain (NTD; residues 1-60) and C-terminal domain (CTD; residues 96-140). To determine fundamental molecular mechanistic aspects of αS function and dysfunction, with emphasis on the dynamics of the IDRs, controlled confinement in a low-temperature, frozen solution system is used to examine the protein-coupled solvent dynamics for monomeric, oligomeric, and fibrillar αS, by using spin-probe (TEMPOL) electron paramagnetic resonance (EPR) spectroscopy. Spin probe and αS forms are colocalized in the ice boundary-delimited interstitial phase. Comparison of αS in oligomeric and fibrillar forms with highly structured soluble globular proteins reveals two major differences: (1) anomalous high fluidity of the αS-coupled solvent under confinement, and (2) compressibility of the protein-coupled solvent disordered regions. Monomeric αS behaves similarly, but the signature thermal hysteresis in phase dynamics and volumes is not observed. These results, augmented by high-resolution structures, lead to an inclusive model, in which the disordered NTD and CTD create a high-fluidity protein-coupled solvent phase with dynamics that persist as the phase volume is decreased by confinement compression. The model is quantified by Arrhenius and van't Hoff analysis, revealing a three-stage confinement response sequence of compaction 1 - consolidation - compaction 2. Effects of cryosolvent addition, varied protein concentration, and domain deletion/truncation on the dynamical properties of αS refine the model. The results and model rationalize the membrane-disrupting properties of cytotoxic αS forms, provide insight into the mechanism of αS function in the crowded neuron presynaptic region, and create a foundation for description of the functional and dysfunctional mechanisms of other IDPs.

Table of Contents

1 Introduction 1

1.1 Intrinsically disordered proteins 1

1.1.1 α-Synuclein 2

1.2 Electron Paramagnetic Resonance 7

1.2.1 History 7

1.2.2 Theory 8

1.3 Mesodomain System 17

1.3.1 Protein Dynamics 17

1.3.2 Controlled, Low-Temperature Confinement System 17

1.3.3 Globular Proteins Under Confinement: The Standard of Protein-Coupled Solvent Dynamics 19

1.4 Spectral Simulation 20

1.5 Overview 22

2 Monomeric, Oligomeric, and Fibrillar α-Synuclein Display Persistent Dynamics and Compressibility Under Strong and Weak Controlled Confinement 24

2.1 Introduction 24

2.2 Experimental Method 25

2.2.1 Sample Preparation 25

2.2.2 EPR measurement 26

2.2.3 EPR simulations 26

2.2.4 TEM 27

2.3 Results 27

2.3.1 Ultrastructure of Monomeric, Oligomeric, and Fibrillar α-Synuclein in EPR Samples Under Strong and Weak Confinement 27

2.3.2 Temperature Dependence of the TEMPOL EPR Spectrum in Frozen Solution Samples of Monomeric, Oligomeric, and Fibrillar α-Synuclein Under Strong and Weak Confinement 29

2.3.3 Temperature Dependence of the TEMPOL Mobilities and Component Weights 34

2.4 Discussion 37

2.4.1 Enhanced Solvent Phase Dynamics around α-Synuclein Forms 37

2.4.2 Cryosolvent-like Solvent Phase Dynamics Are Created by α-Synuclein Oligomers and Fibrils 39

2.4.3 α-Synuclein protein-coupled solvent dynamics in the presence of DMSO 41

2.4.4 Model for Confinement-Resistant Dynamics of the α-Synuclein Forms 42

2.5 Conclusion 45

3 Properties of Confinement-Compaction and Persistent Dynamical Disorder Provides Leads to Molecular Function of Monomeric Alpha-Synuclein 46

3.1 Introduction 46

3.2 Experimental Method 47

3.2.1 Temperature Dependence of Spin Probe Rotational Correlation Time 47

3.2.2 Temperature Dependence of Spin Probe Rotational Correlation Time 47

3.3 Results 48

3.4 Discussion 48

3.4.1 Two compaction transformations and intervening dynamical transition in monomeric α-synuclein are revealed by temperature-controlled confinement 48

3.4.2 Molecular Mechanism of Temperature – Dependent Confinement 52

3.4.3 Thermodynamic Analysis of the Compaction Transition 55

3.4.4 Molecular bases for α-synuclein function in vivo 56

3.5 Conclusion 57

4 Structural Reorganization and Bistability of Fibrillar α-Synuclein Modulated by Confinement 59

4.1 Introduction 59

4.2 Experimental Method 60

4.2.1 Sample Preparation 61

4.2.2 EPR specifications, simulation, and analysis 61

4.3 Results 62

4.3.1 Temperature Dependence of TEMPOL EPR Spectra and Simulated Rotational Correlation Times and Component Weights, Under Different Dimethyl Sulfoxide Concentrations 62

4.3.2 Temperature dependence of TEMPOL spectra and simulated rotational correlation times and component weights, under different fibrillar α-synuclein concentrations 66

4.3.3 Influence of temperature cycling range on thermal hysteresis 68

4.4 Discussion 72

4.4.1 Two distinct compaction transformations characterize the response of fibrillar α-synuclein to increasing confinement 72

4.4.2 Model for fibrillar α-synuclein under temperature-controlled confinement 75

4.4.3 Modulation of confinement by dimethyl sulfoxide 78

4.4.4 Thermal hysteresis arises from bistability in protein and coupled solvent structure 79

4.5 Conclusion 81

5 Truncated Monomeric α-Synuclein Modulated by Confinement 82

5.1 Introduction 82

5.2 Experimental Method 83

5.2.1 Sample Preparation 83

5.2.2 EPR measurement 84

5.3 Results 84

5.3.1 Temperature Dependence of the TEMPOL EPR spectrum in samples of monomeric, truncated α-synuclein 84

5.3.2 Temperature Dependence of the TEMPOL Spin Probe Mobilities and Component Weights 86

5.4 Discussion 88

5.4.1 Common trend of three-stage confinement dependence displayed by full-length and truncated monomeric α-synuclein proteins 88

5.4.2 Inter-domain dynamical effects in full-length and truncated monomeric α-synuclein 89

5.4.3 Consequences of isolated terminal domains in vivo following truncation 90

5.5 Conclusion 90

6 Summary and Conclusions 92

6.1 Overview 92

6.2 Physiological Relevance 93

6.3 Novelty 94

Appendix S1 95

Appendix S2 101

Appendix S3 103

Appendix S4 117

References 121

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School	Laney Graduate School
Department	Physics
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biophysics, General
Keyword	Parkinson's Disease confinement synuclein protein dynamics intrinsically disordered
Committee Chair / Thesis Advisor	Kurt Warncke, Emory University
Committee Members	Justin Burton, Emory University Eric Weeks, Emory University Katherine Davis, Emory University David Lynn, Emory University

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