Influence of epigallocatechin gallate on protein dynamics in oligomeric alpha-synuclein Öffentlichkeit

Lechtzin, Nathaniel (Spring 2023)

Permanent URL: https://etd.library.emory.edu/concern/etds/5999n492s?locale=de
Published

Abstract

The intrinsically disordered protein, -synuclein (-syn) plays a role in neurotransmitter release in brain neurons, and its dysfunction is associated with debilitating neurodegenerative disorders, such as Parkinson's disease. Oligomeric forms of -syn have been identified as cytotoxic, owing to proposed membrane disruption and permeabilization. The small molecule, epigallocatechin gallate (EGCG), a polyphenolic compound derived from green tea, has been shown to bind to oligomeric -syn, remodel oligomer structure, inhibit membrane permeabilization, and suppress the dynamics of intrinsically disordered regions within -syn. Here, we employ temperature-controlled ice boundary confinement and spin probe (TEMPOL) electron paramagnetic resonance spectroscopy in frozen solution samples of -syn in the presence of varying concentrations of EGCG (molar ratios of 1, 7, 15, 60 and 100, relative to -syn) to elucidate the mechanism of action of EGCG. The presence of oligomeric -syn was confirmed in our samples by transmission electron microscopy. Simulation of the electron paramagnetic resonance spectra obtained over the temperature range of 225-265 K reveals two distinct motional components, identified by relatively long (slow rotation) and short (fast rotation) TEMPOL rotational correlation times, and corresponding normalized weights. In the presence of EGCG, the fast component retains the mesophase mobility of -syn alone, independent of EGCG concentration over the full temperature range. In contrast, the slow component mobility decreases for EGCG/-syn ratios greater than 1:1, at each temperature value. The fast component dominance (weight≈0.8) at high temperatures transitions to slow component dominance at low temperatures (>0.9). The crossover temperature of equal weights is shifted to higher temperatures by increasing EGCG concentration. The characteristic thermal hysteresis, which arises from compaction of the dynamics of the disordered C-terminal domain of -syn oligomers is also observed in the presence of EGCG. At high concentrations of EGCG (100:1, 60:1) and elevated temperatures, a redox reaction leads to TEMPOL radical annihilation. Together, the results indicate that EGCG associates predominantly with the N-terminal and central, non-amyloid component of the b-sheet-structured oligomer core, rather than the dynamically disordered C-terminal domain, that protrudes from the core. Overall, we find that EGCG attenuates the dynamics of -syn oligomers globally, making -syn more susceptible to confinement.

Table of Contents

Table of Contents

 

INTRODUCTION                                                                                                                          1

1. The alpha-synuclein protein                                                                                                                  1

2. Aggregate forms of alpha-synuclein                                                                                                      2

3. Effects of the polyphenolic antioxidant, (-)-Epigallocatechin gallate (EGCG), on peptide aggregate structures   3

4. Low-temperature frozen aqueous mesodomain system                                                                      5

5. Spin-probe electron paramagnetic resonance (EPR) spectroscopy                                                     6

6. Prologue                                                                                                                                      11

MATERIALS AND METHODS                                                                                                12

Preparation of EPR samples                                                                                                              12

Continuous-wave EPR spectroscopy                                                                                                  12

Transmission electron microscopy                                                                                                     13

EPR simulations                                                                                                                               13

RESULTS                                                                                                                                     14

1. Temperature dependence of TEMPOL EPR line shape in frozen EGCG solution samples                 14

2. Temperature dependence of TEMPOL EPR line shape in frozen -Syn solution in the presence of varying concentrations of EGCG       16

3. Hysteresis in the Temperature dependence of TEMPOL EPR line shape in frozen -syn solution in the presence of varying concentrations of EGCG      18

4. Redox reaction at elevated EGCG:-Syn and T values leads to TEMPOL radical annihilation           22

5. Simulation of the Temperature dependence of the TEMPOL EPR spectra in the presence of varying concentrations of EGCG    25

6. Temperature dependence of TEMPOL rotational correlation times and normalized component weights under varying EGCG concentrations.     31

7. TEM of aqueous -Syn + EGCG samples.                                                                                      34

DISCUSSION                                                                                                                               36

1. Confirmation of oligomeric -syn by TEM                                                                                     36

2. Analysis of temperature dependence of TEMPOL EPR line shape in frozen EGCG solution sample  36

3. Concentration-dependent effects of EGCG on temperature dependence of TEMPOL EPR line shape in frozen aqueous alpha-synuclein solutions  37

4. Redox reaction of EGCG and alpha-synuclein leads to TEMPOL radical annihilation                                      39

5. Analysis of temperature dependence of TEMPOL rotational correlation times and normalized component weights under varying EGCG concentrations  40

6. Analysis of directional temperature dependence and thermal hysteresis of the TEMPOL EPR spectrum for frozen alpha-synuclein solution in the presence of varying concentrations of EGCG                                                                                                                   42

7. Summary and Conclusions                                                                                                            46

REFERENCES                                                                                                                            49

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