A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures

Abstract

The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to ''burst-phase'' intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was Ϸ43 Å. This increased with denaturant in a noncooperative fashion to Ϸ63 Å, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. (1), involving self-replicating or infectious protein conformations, has attracted broad interest in recent times due to its role in the biology of debilitating neurodegenerative diseases (1, 2), protein-based inheritance of novel phenotypes in yeast (3-5), and (potentially) long-term memory (6, 7). The Saccharomyces cerevisiae translational termination factor, Sup35, is one such protein capable of switching to a self-perpetuating state. In the prion state [PSI ϩ ], the glutamine/asparagine (Q/N)-rich prion domain of Sup35 is sequestered into an amyloid conformer, reducing the efficiencies of translation termination (8). This switch causes ribosomes to read through stop codons at biologically significant rates, changing a multitude of phenotypes (9).The NM segment (253 residues) of Sup35 determines the prion state and comprises two distinct regions. The N-terminal region (residues 1-123) is abundant in uncharged polar amino acids (glutamines, asparagines, and tyrosines), and forms the major part of the amyloid core that directs the protein into the [PSI ϩ ] prion state. The highly charged middle region M (residues 124-250) confers solubility in vitro and in vivo, allowing the protein to exist in the non-prion [psi Ϫ ] state. In the prion state, the N region adopts a ␤-sheet-rich conformation, whereas the M region remains relatively unstructured (10).Structural studies on NM amyloids have provided several insights into the molecular basis of prion nucleation (11)(12)(13)(14). An early step is the establishment of an equilibrium between predominantly unstructured NM polypeptide monomers and molten oligomeric intermediates that are obligate on-pathway species (14-16). The structure and dynamics of early monomeric intermediates are of considerable interest in deciphering the molecular m...

Distinct conformations of in vitro and in vivo amyloids of huntingtin-exon1 show different cytotoxicity

“…This apparent inconsistency of previous results regarding amyloid toxicity may result from the structural diversity of amyloid, as amyloid-forming protein often misfolds into multiple conformations and each conformation could exert distinct physiological effects (19,23). Previous reports indicate that the thermodynamic parameter of temperature can modulate protein folding and dynamics of amyloid-forming proteins, leading to different amyloid conformations (19,(32)(33)(34). Here we took advantages of this fact by making distinct thtt amyloids simply by polymerizing thtt protein at 4°C or 37°C.…”

To fold or expand—a charged question

“…This property keeps them disordered unless they are forced to fold, for example, by binding to a partner protein. However, some IDPs have low charge density but are polar enough to form a loose globular state in the absence of denaturants (6,19). Interestingly, as noted by Müller-Späth et al, even when a protein's chain is electrically neutral overall, a larger number of charged residues may increase its self-attraction and lead to an even more collapsed configuration than dictated by hydrophobic interactions alone.…”

Temperature‐dependent structural changes in intrinsically disordered proteins: Formation of α‒helices or loss of polyproline II?

“…IDPs do not have unique folds but consist of dynamic ensembles of interconverting, intermittent structures. 10,11 The high intramolecular flexibility inherent to IDPs means that distant elements are largely structurally decoupled. Structure formation, and hence function, is thus related to segmental parts of the sequence and dominated by local features.…”