Biomedical subjects
Publications and source records attributed to Ansgar B Siemer.
The application of a spin-state-selective coherence transfer experiment (INADEQUATE-SSS) to solid-state NMR spectroscopy is described. Two-dimensional (13)C double-quantum/single-quantum spectra without J splittings in both dimensions lead to enhanced spectral resolution. The method is demonstrated to significantly improve the spectral resolution of the crowded C'-C(alpha) region of two proteins.
We report the observation of undetected (until now) residues of the prion protein fragment HET-s(218-289) which give rise to well-resolved (13)C, (15)N, and (1)H NMR resonances under high-resolution magic-angle spinning (HRMAS) conditions. The observed signals belong to large polymeric units as shown by measuring the lateral diffusion constants. The amino acids identified in the spectra are compatible with their localization in the segments of the protein which could not be detected in earlier solid-state NMR experiments. The observed chemical shifts indicate that these residues are in a random-coil conformation. Complementary experiments which detect only dynamic or static residues, respectively, strongly suggest that they belong to different parts of the same molecule.
The partial 15N and 13C solid-state NMR resonance assignment of the HET-s prion protein fragment 218-289 in its amyloid form is presented. It is based on experiments measured at MAS frequencies in the range of 20-40 kHz using exclusively adiabatic polarization-transfer schemes. The resonance assignment within each residue is based on two-dimensional 13C-13C correlation spectra utilizing the DREAM mixing scheme. The sequential linking of the assigned residues used a set of two- and three-dimensional 15N-13C correlation experiments. Almost all cross peaks visible in the spectra are assigned, but only resonances from 43 of the 78 amino-acid residues could be detected. The missing residues are thought to be highly disordered and/or highly dynamic giving rise to broad resonance lines that escaped detection in the experiments applied. The line widths of the observed resonances are narrow and comparable to line widths observed in micro-crystalline samples. The 43 assigned residues are located in two fragments of about 20 residues.
Prions are believed to be infectious, self-propagating polymers of otherwise soluble, host-encoded proteins. This concept is now strongly supported by the recent findings that amyloid fibrils of recombinant prion proteins from yeast, Podospora anserina and mammals can induce prion phenotypes in the corresponding hosts. However, the structural basis of prion infectivity remains largely elusive because acquisition of atomic resolution structural properties of amyloid fibrils represents a largely unsolved technical challenge. HET-s, the prion protein of P. anserina, contains a carboxy-terminal prion domain comprising residues 218-289. Amyloid fibrils of HET-s(218-289) are necessary and sufficient for the induction and propagation of prion infectivity. Here, we have used fluorescence studies, quenched hydrogen exchange NMR and solid-state NMR to determine the sequence-specific positions of amyloid fibril secondary structure elements of HET-s(218-289). This approach revealed four beta-strands constituted by two pseudo-repeat sequences, each forming a beta-strand-turn-beta-strand motif. By using a structure-based mutagenesis approach, we show that this conformation is the functional and infectious entity of the HET-s prion. These results correlate distinct structural elements with prion infectivity.
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