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      Diffraction spectroscopy of metalloproteins

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      SHERRELL-DISSERTATION.pdf (48.05Mb)
      Date
      2014-04-02
      Author
      Sherrell, Darren
      Type
      Thesis
      Degree Level
      Doctoral
      Metadata
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      Abstract
      X-ray absorption is not only element specific, but atom specific: two atoms of the same element in different states or in different neighbourhoods will have slightly different absorption characteristics. These energy dependent atomic form factors are carried over to the diffraction intensities. The atomic form factors are sensitive not only to the the energy of the X-ray but also the diffraction criteria; providing individual local physical data at different ratios in various diffractions. This process is referred to as site selectivity, it is unique to Diffraction Spectroscopy, and is achieved only when the sample is in crystal form. Through this work, a technique has been devised to site-separate two atoms of iron from within a protein, that builds on prior small unit cell Diffraction Anomalous Fine Structure experiments and harnesses the collection and processing software commonly used in large unit cell crystallography. A technique (dev + PCA) has been developed to retrieve the small signals from individual atom-labels out of the large and noisy background of real diffraction taken across a spectrum. The intensity of the diffractions are calculated by integrating over multiple images, profiling spots, merging datasets, and scaling across the whole spectrum. This thesis explores how Diffraction Spectroscopy can be used effectively on large unit cells, namely those of proteins. Site-selective absorption experiments were conducted on large unit cell crystals at a 3rd generation beamline, exclusively using existing equipment. The spectra generated were limited in scope but are an adequate proof of concept.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Geological Sciences
      Program
      Biochemistry
      Supervisor
      George, Graham N.; Pickering, Ingrid J.
      Committee
      Chapman, Dean; Merriam, Jim; Szmigielski, Jacek; Grochulski, Pawel; Kennepohl, Pierre
      Copyright Date
      March 2014
      URI
      http://hdl.handle.net/10388/ETD-2014-03-1460
      Subject
      X-ray Crystallography
      X-Ray Absorption Spectroscopy
      Diffraction Anomalous Fine Structure
      Diffraction Spectroscopy
      Metalloprotein
      Redox
      Macromolecule
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