This title appears in the Scientific Report :
2018
Structural characterization of recombinant fibrillar human Islet Amyloid Polypeptide by solid-state Nuclear Magnetic Resonance Spectroscopy
Structural characterization of recombinant fibrillar human Islet Amyloid Polypeptide by solid-state Nuclear Magnetic Resonance Spectroscopy
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 hall...
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Personal Name(s): | Weirich, Franziska (Corresponding author) |
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Contributing Institute: |
Strukturbiochemie; ICS-6 |
Imprint: |
2017
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Physical Description: |
114 p |
Dissertation Note: |
Dissertation, HHU Düsseldorf, 2017 |
Document Type: |
Dissertation / PhD Thesis |
Research Program: |
Physical Basis of Diseases |
Subject (ZB): | |
Publikationsportal JuSER |
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. |