Structural studies of the 75 kDa Sup35 prion: Towards Solid-State NMR for Extra-Large Proteins – XLproteinSSNMR

Solid-state NMR can structurally characterize proteins at different levels. While some years ago, only relatively small proteins were amenable to this technique, today larger protein studies are explored. To give an idea of the sizes and levels proteins can be studied today, one can roughly state that proteins under 100 amino acids are amenable to full high-resolution structure determination by solid-state NMR, as recently shown for different amyloid-beta assemblies. Proteins up to 300-400 amino acids can be assigned sequentially, and thus secondary structure can be accessed. For larger proteins, fingerprints can be recorded, which inform on secondary structure content, order, and on interactions with other molecules. We here extended significantly the last aspect, with the study of the Sup35 prion which is with 685 residues the largest protein studied today by solid-state NMR.

The project yielded atomic-level insight in the structural organization of the Sup35 yeast prion (see Figure). We could show that the protein retains its native fold of its C-terminal domain in the fibrils, and that the first 30 residues build a highly ordered fibril core. Importantly, this core is structurally different when looking at Sup35NM, which is devoid of the C-terminal globular domain. This speaks against the use of Sup35NM as a faithful model for the study of yeast prion biology. We could also show for other large assemblies, as molecular motor proteins and protein fibrils, how they are organized and interact with partners.

Our studies open the way for other complex assemblies.

We have published 27 papers in diverse journals, ranging from specialized to general, according to the results obtained.

Little is known today about the structure of full-length prions. We recently established, using solid-state NMR studies of the Ure2p and HET-s proteins, that prions show different architectures, and that the building principles between them can differ considerably. We here aim at the study of the Sup35 prion, which poses a formidable challenge considering its large size. Having demonstrated the important role of the globular domain, we here aim to study assembly in the context of the entire proteins, as opposed to fragments, and investigate on a molecular level the role of the functional C-terminal domain of the protein in fibril assembly. We aim thus to compare the structures of fibrillar Sup35NM and full-length Sup35p by solid state NMR. The fragment Sup35NM is often used as a model for the full-length prion. We will compare the structures of fibrillar Sup35NM and full-length Sup35p by solid state NMR and determine whether the structure of this fragment is indeed preserved in the context of the full-length fibril, or if it is different. This question is particularly relevant in the light of our previous results on the full-length HET-s and Ure2p prions, which show that the structure of the isolated prion and globular domains are not conserved in the context of the full-length prions.
We will address this question by solid-state NMR, which is an emerging technique for the structural study of insoluble proteins. Important developments over the last decade have pushed this fast evolving technique to become a serious partner in structural biology. If it has been shown in proof-of-principle experiments that amyloid and prion structures can be determined by solid-state NMR methods, it is still difficult to obtain structures of full-length prions. The main difficulties are caused by the large size of these proteins, a property they also share with other insoluble proteins. However, many elements of the technology needed to tackle these problems are now available in the applicants’ laboratories and we propose to fully develop the NMR methodology and, in parallel, apply it to the yeast prion Sup35p that is made of 685 amino-acid residues.
The key NMR methodology developed in the context of this proposal will allow us to establish structural models which will be confronted with the large body of biophysical and biochemical and functional knowledge to work out the relationship between structural and biological features of Sup35p. Besides, the protocols and techniques developed will also be applicable to other large proteins and should allow to open structural studies on other insoluble proteins.