Mass Spectroscopic Analysis of Sup35NM Prion Polymerization

Abstract

Sup35NM, the prion determining domain of the protein responsible for the yeast prion phenomenon [Psi], has become a powerful model for studying key processes in amyloid-related human diseases. One of these processes is a conformational conversion of soluble precursor protein into insoluble fibrillar structures. In this study, we created a set of Sup35NM mutants and used proteolytic digestion coupled with mass spectroscopy to monitor local structure of the protein during polymerization. Experimental data were compared to a network model and showed that during the conformational conversion residue Arg-28 became highly protected from cleavage, residue Arg-98 remained partially solvent exposed, and residues between 28 and 98 showed an intermediate degree of protection. In addition, we found that a distinct subset of proteolytic polypeptides spanning 28-98 residues segment spontaneously formed stable dimers. This finding suggests that the [29-98] region is the key interacting region of Sup35NM responsible for amyloid conversion.

Temperature Dependence of the Aggregation Kinetics of Sup35 and Ure2p Yeast Prions

“…The Sup35 NM (1–254) and Het-s PFD (218–289) regions typically used to study the aggregation and infective properties of the correspondent proteins correspond to the prion-forming domains of these molecules and are likely devoid of any significant regular secondary or tertiary structure in their respective monomeric states. − In contrast, the full-length protein, comprising both the prion unstructured region and a globular α-helical domain displaying a glutathione-S-transferase-like fold, has been used traditionally to study the aggregation and transmissibility of Ure2p, as is the case of the present study. Energetic calculations suggest that the nucleation step of Ure2p amyloid formation involves a structural rearrangement of the molecule.…”

Protonated Arginine and Protonated Lysine: Hydration and Its Effect on the Stability of Salt-Bridge Structures

“…Arginine (Arg) and lysine (Lys), two of the 20 naturally occurring amino acids, have basic side chains with the potential to be protonated and carry a positive charge. In the field of mass spectrometry (MS), these two amino acids play a crucial role in protein sequencing and conformational analysis techniques ranging from collision-induced dissociation (CID), surface-induced dissociation (SID), and electron-capture dissociation (ECD) to proteolytic digestion analyzed by MS. − For instance, an MS study on the Sup35NM prion domain sequence employing proteolytic digestion protocols, which target arginine as cleavage sites, provides conformational information on this protein relevant with respect to prion fibril formation. Another example is a study on the stability of gas-phase helices, where the position of protonated lysine in the peptide sequence is found to be the main criterion controlling the helix stability. , …”