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University of Guelph

1. Ward, Meaghan Elizabeth. Solid-State NMR Investigations of Transmembrane Proteins - New Approaches for Signal Enhancement and In Situ Studies of Anabaena Sensory Rhodopsin.

Degree: PhD, Department of Physics, 2016, University of Guelph

Membrane proteins account for 30% of all proteins and perform many important roles in the cell, yet are difficult to study due to the necessity to maintain their lipidic environment during all stages of purification and characterization. Often during these processes the lipid environment is drastically altered, and high resolution studies of membrane protein structure commonly require the use of membrane-mimetic environments. Solid-state NMR (SSNMR) spectroscopy using magic angle spinning (MAS) is a rapidly developing and promising technique to study such proteins in their native, lipid-associated environment. However, these experiments are generally limited by sensitivity. This thesis centers upon the development and application of novel SSNMR approaches to study large, seven transmembrane (7TM) α-helical proteins – a class of proteins to which microbial rhodopsins, which we use as model systems for these experiments, belong. First, the sensitivity available for such samples under ultrafast MAS conditions (> 50 kHz) was investigated. As smaller sample volumes are necessary at these spinning frequencies, low power decoupling and paramagnetic enhancement of the signal relaxation rate were implemented to facilitate a condensed data collection scheme. Under these conditions, it was found that the paramagnetic relaxation enhancement was uniformly distributed throughout the proteins and that sensitivity comparable to that available in larger rotors was obtainable with proton detection. Next, the implementation of proton detection to specifically detect the mobile regions of proteins was developed. Using these methods it was found that for the mobiles regions, ~10x increase in sensitivity was available and that both the loop regions and lipid and carbohydrates tightly bound to these proteins could be studied. Finally, methods with which to characterize membrane proteins in the native E. coli membrane environment were developed and implemented, using Anabaena sensory rhodopsin (ASR) as an example. Small, site-specific perturbations in the structure of ASR, which occur as the local membrane milieu changes, indicate that the protein can subtly adapt to its environment without large structural rearrangement. In summary, this work has advanced our ability to use MAS-SSNMR spectroscopy as a structural probe for large and oligomeric membrane proteins. Advisors/Committee Members: Ladizhansky, Vladimir (advisor), Brown, Leonid (advisor).

Subjects/Keywords: solid state nuclear magnetic resonance; membrane protein; proton detection; In situ; microbial rhodopsin; magic angle spinning; paramagnetic relaxation; condensed data collection

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Ward, M. E. (2016). Solid-State NMR Investigations of Transmembrane Proteins - New Approaches for Signal Enhancement and In Situ Studies of Anabaena Sensory Rhodopsin. (Doctoral Dissertation). University of Guelph. Retrieved from https://atrium.lib.uoguelph.ca/xmlui/handle/10214/9784

Chicago Manual of Style (16th Edition):

Ward, Meaghan Elizabeth. “Solid-State NMR Investigations of Transmembrane Proteins - New Approaches for Signal Enhancement and In Situ Studies of Anabaena Sensory Rhodopsin.” 2016. Doctoral Dissertation, University of Guelph. Accessed October 31, 2020. https://atrium.lib.uoguelph.ca/xmlui/handle/10214/9784.

MLA Handbook (7th Edition):

Ward, Meaghan Elizabeth. “Solid-State NMR Investigations of Transmembrane Proteins - New Approaches for Signal Enhancement and In Situ Studies of Anabaena Sensory Rhodopsin.” 2016. Web. 31 Oct 2020.

Vancouver:

Ward ME. Solid-State NMR Investigations of Transmembrane Proteins - New Approaches for Signal Enhancement and In Situ Studies of Anabaena Sensory Rhodopsin. [Internet] [Doctoral dissertation]. University of Guelph; 2016. [cited 2020 Oct 31]. Available from: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/9784.

Council of Science Editors:

Ward ME. Solid-State NMR Investigations of Transmembrane Proteins - New Approaches for Signal Enhancement and In Situ Studies of Anabaena Sensory Rhodopsin. [Doctoral Dissertation]. University of Guelph; 2016. Available from: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/9784


University of Illinois – Chicago

2. Parthasarathy, Sudhakar. Solid-State Nuclear Magnetic Resonance of Copper-Amyloid Beta, Amylospheroids, Fast Magic Angle Spinning.

Degree: 2013, University of Illinois – Chicago

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. Advisors/Committee Members: Ishii, Yoshitaka (advisor), Fung, Leslie (committee member), Keiderling, Timothy (committee member), Lorieau, Justin (committee member), Ramirez, Benjamin (committee member).

Subjects/Keywords: Alzheimer’s Disease; Amyloid beta; Amylospheroids; Copper-Amyloid beta; Ascorbate; Hydrogen Peroxide; Reactive Oxygen Species; Oxidative Stress; Paramagnetic Relaxation Assisted Condensed Data Collection; Solid-State Nuclear Magnetic Resonance; Ultra-fast Magic Angle Spinning

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Parthasarathy, S. (2013). Solid-State Nuclear Magnetic Resonance of Copper-Amyloid Beta, Amylospheroids, Fast Magic Angle Spinning. (Thesis). University of Illinois – Chicago. Retrieved from http://hdl.handle.net/10027/10058

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Parthasarathy, Sudhakar. “Solid-State Nuclear Magnetic Resonance of Copper-Amyloid Beta, Amylospheroids, Fast Magic Angle Spinning.” 2013. Thesis, University of Illinois – Chicago. Accessed October 31, 2020. http://hdl.handle.net/10027/10058.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Parthasarathy, Sudhakar. “Solid-State Nuclear Magnetic Resonance of Copper-Amyloid Beta, Amylospheroids, Fast Magic Angle Spinning.” 2013. Web. 31 Oct 2020.

Vancouver:

Parthasarathy S. Solid-State Nuclear Magnetic Resonance of Copper-Amyloid Beta, Amylospheroids, Fast Magic Angle Spinning. [Internet] [Thesis]. University of Illinois – Chicago; 2013. [cited 2020 Oct 31]. Available from: http://hdl.handle.net/10027/10058.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Parthasarathy S. Solid-State Nuclear Magnetic Resonance of Copper-Amyloid Beta, Amylospheroids, Fast Magic Angle Spinning. [Thesis]. University of Illinois – Chicago; 2013. Available from: http://hdl.handle.net/10027/10058

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

.