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.

FIGURE 1

Digestion network model. Figure shows schematics of proteolytic digestion for a protein with two cleavage sites. Proteolysis of wild-type Sup35NM by Arg-C protease is described by this model. Rectangles represent different proteolytic peptides liberated during the digestion. Bended arrows represent the graph's edges. They signify cleavage pathways. Large light gray circles represent nodes of the graph. The intensities of the nodes correspond to the time-dependent normalized intensities of the proteolytic fragments X_ij. They are calculated as a solution to Eq. 1. The solution depends on S₁ and S₂—cleavage rates at proteolytic sites 1 and 2. The cleavage rates are determined by minimizing Eq. 2.

FIGURE 2

Sup35NM Arg-C digestion spectra. MALDI spectra of Sup35NM digested with Arg-C protease. The fiber formation reactions were initiated by addition of seed and then frozen at various stages of polymerization. (A) No seed added; (B) 1 min after addition of seed; (C) 30 min; (D) 60 min; (E) 120 min. All samples were digested simultaneously, desalted and analyzed by MALDI.

FIGURE 3

Sup35RM EM images. Electron micrographs of the negatively stained seeded fiber samples: Sup35NM-G7R, Sup35NM-N21R, Sup35NM-G51R, Sup35NM-G58R, Sup35NM-G68R, and Sup35NM-G96R. All proteins assemble into fibers with similar morphology. Scale bar, 100 nm.

FIGURE 4

Sup35RM thioflavin-T kinetics. (A) Unseeded kinetics. Soluble protein samples were mixed with TfT and gently stirred during the course of reaction. The changes in TfT fluorescence occurs when TfT binds to the fibers. All proteins except Sup35NM-G7R show unseeded kinetics similar to the wild-type Sup35NM. Sup35NM-G7R has lag phase ∼40% larger than wild type. (B) Seeded kinetics. Seeded polymerization reactions were monitored by TfT binding. All proteins except Sup35NM-N21R show kinetics similar to wild-type Sup35NM. N21R mutant has slower apparent assembly rates than the wild type.

FIGURE 5

Sup35RM digestion spectra MALDI spectra of initial no seed (left) and fibrous (right) samples digested with Arg-C protease. Relative intensities of the fragments change significantly during the course of fiber formation. Spectra of initial samples is dominated by completely cut end-fragments, whereas “120 min” fiber samples show significant presence of undigested fragments containing one or two cleavage sites.

FIGURE 6

Sup35 peptide dimers MALDI spectra show the presence of stable dimers formed from the proteolytic peptides. Dimers are formed only if both peptides span the residue subset from [29–98]. Thus, homodimers [2–98]², [29–98]² and heterodimers [29–98] × [2–98] are formed, but not [2–28] × [2–98] or [2–28]².

FIGURE 7

Interacting regions of Sup35NM. At the top of the figure, intensity of black color corresponds to the region's propensity to dimerize. (Bottom) Solid black rectangles at the bottom of the figure signify the peptides that formed dimers; light gray rectangles signify the peptides that did not form dimers.