Influence of epigallocatechin gallate on protein dynamics in oligomeric alpha-synuclein Público
Lechtzin, Nathaniel (Spring 2023)
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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