Parthasarathy_Sudhakar.pdf (7.63 MB)
Solid-State Nuclear Magnetic Resonance of Copper-Amyloid Beta, Amylospheroids, Fast Magic Angle Spinning
thesisposted on 2013-06-28, 00:00 authored by Sudhakar Parthasarathy
Molecular level structural examination of Cu2+ binding to 40-residue Alzheimer’s amyloid β (Aβ(1-40)) peptides and the resultant production of reactive oxygen species (ROS) was studied by SSNMR and other techniques. Aβ(1-40) peptide is one of the primary component of senile plaques in Alzheimer’s disease (AD) and was shown that Cu ions (400 µM) are accumulated in plaque deposits and bind Aβ peptides. The Cu-Aβ complex is believed to trigger the production of ROS causing oxidative stress. Despite oxidative stress being considered one of the probable mechanisms of AD, detailed binding structure of Cu2+-Aβ fibrils and the molecular level mechanism of the production of ROS in presence of cellular reductants are still largely unknown. In our work, we try to understand the molecular level details of Cu2+ binding to Aβ(1-40) fibrils, the mechanism of ROS production and subsequent binding of Cu ions with Aβ(1-40) fibrils. Initial signal quenching studies by 13C SSNMR and molecular dynamics simulations showed Cu2+ binding to Aβ(1-40) fibrils at Nε in His-13, His-14 and carboxyl groups in Val-40 as well as Glu sidechains (Glu-3,Glu-11, and/or Glu-22). In the presence of physiological amounts of biological reductant ascorbate Cu2+-Aβ(1-40) complex generates ~60 μM H2O2. During the production of H2O2, Cu2+-Aβ(1-40) fibrils undergoes cyclic redox reaction (Cu2+ ↔ Cu+ )- Aβ(1-40) without any alteration to the fibrils. SSNMR experiments revealed that Cu+ ions are bound to Nδ in His-13 and His-14 during the ROS production. In the second part, the site-specific molecular level structural features of the synthetic Aβ(1-42) amylo-spheroids (ASPD) which is a structural/morphological analog of native ASPD extracted from AD patients is discussed. ASPD represents a class of highly cytotoxic and high mass (>100 kDa) spherical aggregates which have distinct surface tertiary structure and induce degeneration of mature neurons through a different mechanism from other non-fibrillar Aβ assemblies and considered to play a primary role in AD. In the last part, sensitivity and resolution enhancement in 13C and 1H SSNMR is demonstrated by paramagnetic relaxation assisted condensed data collection method, ultra-fast MAS (≥ 78kHz) and selective deuteration using stereo array isotope labeling (SAIL) in a high magnetic field.
Degree GrantorUniversity of Illinois at Chicago
Committee MemberFung, Leslie Keiderling, Timothy Lorieau, Justin Ramirez, Benjamin