Prion Amyloid Polymorphs - The Tag Might Change It All.

Sup35p is a protein from Saccharomyces cerevisiae. It can propagate using a prion-like mechanism, which means that it can recruit non-prion soluble Sup35p into insoluble fibrils. Sup35p is a large protein showing three distinct domains, N, M and an extended globular domain. We have previously studied the conformations of the full-length and truncated NM versions carrying poly-histidine tags on the N-terminus. Comparison with structural data from C-terminally poly-histidine tagged NM from the literature surprisingly revealed discrepancies. Here we investigated fibrils from the untagged, as well as a C-terminally poly-histidine tagged NM construct, using solid-state NMR. We find that the conformation of untagged NM is very close to the N-terminally tagged NM and confirms our previous findings. The C-terminal poly-histidine tag, in contrast, drastically changes the NM fibril structure, and yields data consistent with results obtained previously on this construct. We conclude that the C-terminally located Sup35p globular domain influences the structure of the fibrillar core at the N domain, as previously shown. We further conclude, based on the present data, that small tags on NM C-terminus have a substantial, despite different, impact. Modifications at this remote localization thus shows an unexpected influence on the fibril structure, and importantly also its propensity to induce [PSI+].

Solid-state NMR sequential assignments of the C-terminal oligomerization domain of human C4b-binding protein.

The complement 4 binding protein (C4bp) plays a crucial role in the inhibition of the complement cascade. It has an extraordinary seven-arm octopus-like structure with 7 tentacle-like identical chains, held together at their C-terminal end. The C-terminal domain does oligomerize in isolation, and is necessary and sufficient to oligomerize full-length C4bp. It is predicted to form a seven-helix coiled coil, and its multimerization properties make it a promising vaccine adjuvant, probably by enhancing the structural stability and binding affinity of the presented antigen. Here, we present the solid-state NMR resonance assignment of the human C4bp C-terminal oligomerization Domain, hC4pbOD, and the corresponding secondary chemical shifts.

Solid-state NMR sequential assignments of the amyloid core of full-length Sup35p.

Sup35p is a yeast prion and is responsible for the [PSI(+)] trait in Saccharomyces cerevisiae. With 685 amino acids, full-length soluble and fibrillar Sup35p are challenging targets for structural biology as they cannot be investigated by X-ray crystallography or NMR in solution. We present solid-state NMR studies of fibrils formed by the full-length Sup35 protein. We detect an ordered and rigid core of the protein that gives rise to narrow and strong peaks, while large parts of the protein show either static disorder or dynamics on time scales which interfere with dipolar polarization transfer or shorten the coherence lifetime. Thus, only a small subset of resonances is observed in 3D spectra. Here we describe in detail the sequential assignments of the 22 residues for which resonances are observed in 3D spectra: their chemical shifts mostly corresponding to ß-sheet secondary structure. We suspect that these residues form the amyloid core of the fibril.

Solid-state NMR sequential assignments of the amyloid core of Sup35pNM.

Sup35pNM represents the N-terminal and middle (M) domains of the yeast Saccharomyces cerevisiae prion Sup35p. This fragment is commonly used for structural and functional studies of Sup35p. We here present a solid-state NMR study of fibrils formed by this fragment and show that sequential assignments can be obtained for the rigid and well-ordered parts of the protein using 3D spectroscopy. We describe in detail the sequential assignment of the 22 residues yielding strong, narrow signals with chemical shifts that correspond mostly to ß-sheet secondary-structured amino acids that form the fibril core.