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...

Single-molecule methods have matured into powerful and popular tools to probe the complex behaviour of biological molecules, due to their unique abilities to probe molecular structure, dynamics and function, unhindered by the averaging inherent in ensemble experiments. This review presents an overview of the burgeoning field of single-molecule biophysics, discussing key highlights and selected examples from its genesis to our projections for its future. Following brief introductions to a few popular single-molecule fluorescence and manipulation methods, we discuss novel insights gained from single-molecule studies in key biological areas ranging from biological folding to experiments performed in vivo.

We have investigated the fibrillation propensity of different conformational isomers of an archetypal, all α-helical protein, namely, bovine serum albumin (BSA), under different pH conditions and ionic strengths using fluorescence and circular dichroism (CD) spectroscopy. At low pH and higher protein concentration, the partially folded conformers associate to form oligomers that are converted into ordered amyloid-like fibrils when incubated at elevated temperature. We have elucidated the mechanism of fibril formation, especially the early steps, by monitoring the kinetics of structural changes during the aggregation process. Various structural probes in tandem were utilized to decipher the temporal evolution of both conformational and size changes by measuring the time dependence of fluorescence intensity and anisotropy of intrinsic tryptophans and several extrinsic fluorophores during the aggregation. Additionally, CD spectroscopy was utilized to monitor the changes in protein secondary structural content during fibrillation. Our findings suggest that the conformational conversion occurs in the oligomers that serve as precursors to amyloid fibrils and precedes the overall fibrillar growth.

Some amyloid-forming polypeptides are associated with devastating human diseases and others provide important biological functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign versus toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the priondetermining region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small molecules or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Aβ, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Aβ, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike.

MaterialsAll of the chemicals used for preparing buffer solutions, such as, sodium phosphate monobasic dihydrate, Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), MES (2-(N-Morpholino)ethanesulfonic acid hydrate, EDTA (Ethylenediaminetetraacetic acid), Magnesium chloride hexahydrate, DTT (DL-Dithiothreitol) were of highest purity grade obtained from Sigma Aldrich (St. Louis, MO). The fluorescent probes, namely, fluorescein-5maleimide, N-(1-pyrene) maleimide, Acrylodan (6-Acryloyl-2-Dimethylaminonaphthalene), AlexaFluor 488 C5-maleimide, and AlexaFluor 594 C5-maleimide were purchased from Molecular Probes, Invitrogen. The free fluorescein dye was purchased from Fluka Analytical. SP Sepharose resin used for protein purification and PD-10 columns were purchased from GE Healthcare Life Sciences (USA). The protein concentrators and filters were procured from Merck Millipore. A Metrohm 827 lab pH meter was used to adjust the final pH ( 0.01) of all the buffer solutions prepared in Milli-Q water and filtered before use. Expression and Purification of tau K18Tau K18 was expressed in Escherichia coli BL21(DE3) and purified using the procedure described previously (51). Briefly, using a lysis buffer of pH 8, (50 mM Tris, 150 mM NaCl, 10 mM EDTA), the cells were lysed by boiling it for half an hour at a 100 ˚C. The lysate was then centrifuged at 11,500 rpm at 4 ˚C for 30 min, following which the supernatant was treated with 136 µL/mL of 10% streptomycin sulfate and 228 µL/mL of glacial acetic acid for the precipitation of DNA. After the removal of DNA by further centrifugation at 11,500