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@PHDTHESIS{Weirich:836105,
      author       = {Weirich, Franziska},
      title        = {{S}tructural characterization of recombinant fibrillar
                      human {I}slet {A}myloid {P}olypeptide by solid-state
                      {N}uclear {M}agnetic {R}esonance {S}pectroscopy},
      school       = {HHU Düsseldorf},
      type         = {Dissertation},
      reportid     = {FZJ-2017-05229},
      pages        = {114 p},
      year         = {2017},
      note         = {Dissertation, HHU Düsseldorf, 2017},
      abstract     = {In this thesis, the method of solid-state Nuclear Magnetic
                      Resonance spectroscopy was applied to solve a specific
                      question from the field of protein misfolding diseases.
                      Representatives of protein misfolding diseases are diabetes
                      mellitus type II, Alzheimer´s disease, and Parkinson´s
                      disease. A hallmark of protein misfolding diseases is the
                      extracellular deposition of misfolded proteins, either
                      localized in affected tissue, or systemic throughout the
                      body. The process of protein misfolding leads in many cases
                      to the formation of insoluble amyloid fibrils. Amyloid
                      fibrils formed by a range of different proteins show a
                      highly ordered and repetitive structure, which is rich in
                      β-sheet content. Elucidation of fibril structures on the
                      molecular level is one of the central research topics in the
                      field of protein misfolding diseases.The topic of this
                      dissertation was the structural characterization of
                      fibrillar human Islet-Amyloid-Polypeptide (IAPP) by
                      solid-state NMR. IAPP is a 37 amino acid residue peptide
                      that is cosecreted with insulin by pancreatic β-cells. IAPP
                      is soluble and intrinsically disordered in its physiological
                      form, however it shows a high propensity to aggregation and
                      fibril formation. Pancreatic amyloid deposits, which consist
                      mainly of IAPP, correlate with type II diabetes mellitus and
                      are found in $90\%$ of individuals affected by T2DM. Human
                      IAPP has an oxidized disulfide bridge in its N-terminus and
                      an amidated C-terminus. In this thesis, recombinantly
                      expressed IAPP with an intact disulfide bridge, but without
                      an amidated C-terminus, was used. It is denoted as
                      IAPPCOOH.Solid-state NMR is a versatile tool to study the
                      structure of fibrils built from full length proteins. These
                      samples are not amenable to X-ray crystallography or
                      liquid-state NMR, because they are non-crystalline and
                      insoluble. Solid-state NMR studies on biomolecules are based
                      on the NMR active isotopes 1H, 13C, and 15N, employing
                      isotope labelling strategies. Information on secondary and
                      tertiary structure is contained in chemical shifts and
                      strengths of dipolar couplings. A full site-specific
                      resonance assignment was achieved from the ssNMR study on
                      fibrillar IAPP. An analysis of secondary structure was
                      performed based on the chemical shifts. One major
                      conformation was observed, consisting of three β-strands.
                      The amyloidogenic segment 22NFGAILS28was also found to be
                      part of a β-strand. Furthermore, the N-terminus showed well
                      resolved and intense peaks at 0°C, that broadened below
                      detectability upon freezing to -170°C. These results have
                      been published in the journal PLoS One in 2016. Following
                      experiments aimed at the detection of through-space dipolar
                      couplings, which contain information on the tertiary
                      structure of the molecules within the fibril. Two more
                      samples were prepared, applying a labelling approach that
                      yields a diluted distribution of 13C spins in the samples.
                      The dilution of NMR-active spins enables the detection of
                      weak through-space dipolar couplings, which are attenuated
                      in uniformly labelled samples by the effect of dipolar
                      truncation. Complementary, long mixing time 13C-13C
                      correlation experiments were performed on a diluted (1:4)
                      and an uniformly labelled sample. Four ambiguous long-range
                      cross-peaks were observed in these spectra. They could
                      contain information on the tertiary fold of the molecules
                      within the fibrils. Four ambiguous distance restraints were
                      created from these cross-peaks and used in a first structure
                      calculation with the computer program Cyana. Moreover, based
                      on the analysis of secondary structure and the varying
                      intensity of Cα-Cβ crosspeaks in short mixing time PDSD
                      spectra, two more structural models were created.},
      cin          = {ICS-6},
      cid          = {I:(DE-Juel1)ICS-6-20110106},
      pnm          = {553 - Physical Basis of Diseases (POF3-553)},
      pid          = {G:(DE-HGF)POF3-553},
      typ          = {PUB:(DE-HGF)11},
      url          = {https://juser.fz-juelich.de/record/836105},
}