School of Physics - Theses

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    Structure of Zinc and Metal Binding Sites in N-truncated Cu Amyloid-beta from X-ray Absorption Fine Structure
    Ekanayake, Ruwini Supeshala Kumari ( 2021)
    X-ray absorption spectroscopy (XAS) is an advanced technique to explore structural information of many types of materials due to its sensitivity and adaptability. Quantum interference of the incoming and outgoing photoelectrons of an absorbing atom reveals the local environment of the absorbing atom and hence a number of fundamental parameters of the material. However, detection of high accuracy data and propagation of experimental uncertainties is limited due to lack of technology. This thesis implemented high accuracy techniques such as the X-ray extended range technique (XERT) and the Hybrid technique to collect high accuracy absorption and fluorescence measurements at ambient temperatures. Protocols of these techniques dictate that systematic errors are investigated over an extended range of experimental parameter space, resulting in better analysis of the mass attenuation coefficients and X-ray absorption fine structure (XAFS) across the K-edge. These techniques can be used to investigate significant systematic errors such as dark currents, blank normalization, harmonics, scattering effects, thickness effect, energy bandwidth, roughness and energy calibration and correct them for precise extended X-ray absorption fine structure (EXAFS) analysis of zinc. The first XERT-like experiment at the Australian Synchrotron was successfully implemented on the XAS beamline to collect high accurate X-ray mass attenuation coefficients across an energy range including the zinc K-absorption edge and XAFS of zinc. Dark current correction was quantified and reached up to 57% for thicker foils and was also significant for thin sample foils. Blank measurements normalized attenuation measurements and scaled thin foil attenuation by 60-500% and even corrected thicker foil attenuation by up to 90%. Discrepancies between different thick foils of up to 20% is corrected using the full-foil mapping technique. The energy was calibrated using standard reference foils. Fluorescence scattering was significant for these measurements and explored carefully. A method base on the different aperture combination was introduced to investigate fluorescence radiation. In this current work, fluorescence radiation has a large impact on the attenuation measurements of thicker sample foils. The correction is energy and sample thickness dependent and therefore significantly affected on the oscillations in the near-edge region. The occurrence of background fluorescence scattering from an unidentified background object in the upstream beamline was observed and corrected for zinc measurements. The correction of fluorescence radiation changes the attenuation of measurements by up to 15.5% and reduced the standard error from the dispersion and the variance by up to 50.0% for thickest sample foil. These results produce the most accurate mass attenuation measurements of zinc from 34.77 to 323.76 (cm2g-1) over the energy range from 8.51 keV to 11.59 keV. The absolute experimental uncertainties were propagated based on systematics and range from 0.023% to 0.036%. These high accuracy studies enable rigorous investigations of discrepancies between theory and experiment and precise structural investigations. The experimentally obtained mass attenuation coefficients deviate by abut 50% from the theoretically tabulated vales near the zinc K-edge. This strongly implies the improvements in theoretical tabulations of the mass attenuation coefficients. The high accuracy data for zinc led to the ability to derive the imaginary component of the atomic form factors and a novel investigation of edge factor and edge ratio of zinc. The XAFS analysis yielded bond lengths and nanostructure of zinc with uncertainties from 0.003 angstrom to 0.008 angstrom. This is superior to many crystallographic analyses of spacing from lattice structure, and is sufficient to investigate and determine thermal parameters with an accuracy of 5%. Our high accuracy data provide great insights into local dynamic motion that is impossible to observe through conventional crystallography. These results can be used for explicit explorations of solid-state effects including inelastic mean free paths, inelastic and elastic scattering cross-section. XAS is an ideal, element selecting tool to investigate many biological samples such as organometals and metal peptides as it provides high resolution structural information and is suitable for sensitive samples. N-truncated Cu:Amyloid-beta (Cu:Abeta) peptide complex contributes to oxidative stress and neurotoxicity in Alzheimer's patient's brains. Redox properties of Cu metal in different Amyloid-beta peptide sequences are inconsistent. Our novel X-ray absorption spectroscopy spectro-electrochemical technique (XAS-SEC) allows an understanding of redox characteristics of Cu ion in different Cu:Abeta peptide sequences and the structural information such as bond lengths and thermal parameters of Cu metal binding sites under near physiological conditions. We determined the geometry of binding sites for the key Cu binding in Abeta4-9/12/16 and the ability of these peptides to perform redox cycle in a manner that might produce toxicity in human brains. We propagated experimental uncertainties due to systematics errors and incorporated in EXAFS analysis for determining precise structural parameters with reliable uncertainties. Our low temperature XAS measurements reveal that Cu(II) is bound to the first amino acids, in the high-affinity amino-terminal copper nickel (ATCUN) binding motif, with an oxygen in a tetragonal pyramid geometry in the Abeta4-9/12/16 peptides. Room temperature XAS-SEC measurements implies metal reduction in Abeta4-16 peptide. Robust investigations of EXAFS provide structural details of Cu(I) binding with bis-His motif and a water oxygen in quasi tetrahedral geometry. Oxidized XAS measurements of Abeta4-12/16 reveal that both Cu(II) and Cu(III) are accommodated in ATCUN-like binding site. A new protocol was developed using EXAFS data analysis for monitoring radiation damage.