Dominic Narang

Nanoscale Fluorescence Imaging of Single Amyloid Fibrils.

Amyloid formation is implicated in a variety of human diseases. It is important to perform high-resolution optical imaging of individual amyloid fibrils to delineate the structural basis of supramolecular protein assembly. However, amyloid fibrils do not lend themselves to the conventional microscopic resolution, which is hindered by the diffraction limit. Here we show super-resolution fluorescence imaging of fluorescently stained amyloid fibrils derived from disease-associated human β2-microglobulin using near-field scanning fluorescence microscopy. Using this technique, we were able to resolve the fibrils that were spatially separated by ∼75 nm. We have also been able to interrogate individual fibrils in a fibril-by-fibril manner by simultaneously monitoring both nanoscale topography and fluorescence brightness along the length of the fibrils. This method holds promise to detect conformational distributions and heterogeneity that are believed to correlate with the supramolecular packing of misfolded proteins within the fibrils in a diverse conformationally enciphered prion strains and amyloid polymorphs.

Characterization of Salt-Induced Oligomerization of Human β2-Microglobulin at Low pH.

Misfolding and amyloid aggregation of human β2-microglobulin (β2m) have been linked to dialysis-related amyloidosis. Previous studies have shown that in the presence of different salt concentrations and at pH 2.5, β2m assembles into aggregates with distinct morphologies. However, the structural and mechanistic details of the aggregation of β2m, giving rise to different morphologies, are poorly understood. In this work, we have extensively characterized the salt-induced oligomers of the acid-unfolded state of β2m using an array of biophysical tools including steady-state and time-resolved fluorescence, circular dichroism, dynamic light scattering, and atomic force microscopy imaging. Fluorescence studies using the oligomer-sensitive molecular rotor, 4-(dicyanovinyl)-julolidine, in conjunction with the light scattering and cross-linking assay indicated that at low salt (NaCl) concentrations β2m exists as a disordered monomer, capable of transforming into ordered amyloid. In the presence of higher concentrations of salt, β2m aggregates into a larger oligomeric species that does not appear to transform into amyloid fibrils. Site-specific fluorescence experiments using single Trp variants of β2m revealed that the middle region of the protein is incorporated into these oligomers, whereas the C-terminal segment is highly exposed to bulk water. Additionally, stopped-flow kinetic experiments indicated that the formation of hydrophobic core and oligomerization occur concomitantly. Our results revealed the distinct pathways by which β2m assembles into oligomers and fibrils.

Site-Specific Fluorescence Depolarization Kinetics Distinguishes the Amyloid Folds Responsible for Distinct Yeast Prion Strains

The prion determinant of a yeast prion protein, Sup35NM, assembles into β-rich amyloid fibrils that switch the nonprion [psi-] state to the prion [PSI+] state of yeast. Previous studies showed that two distinct forms of amyloids (Sc4 and Sc37), generated in vitro at two different temperatures (4 and 37 °C), recapitulate the strain phenomenon in Saccharomyces cerevisiae. Sc4 demonstrates a strong [PSI+] phenotype, whereas Sc37 shows a weak phenotype. To discern the residue-specific structural and dynamical attributes associated with the amyloids that display strain diversity, we took advantage of the nonoccurrence of tryptophan (Trp) in the NM-domain and created 18 single-Trp variants spanning the entire polypeptide length. The fluorescence readouts from these locations reported the site-specific structural details in Sc4 and Sc37 fibrils. Highly sensitive picosecond fluorescence depolarization measurements at these positions allowed a conformational mobility map to be constructed. Nearly all of the residue positions demonstrated higher local flexibility in Sc4 amyloid, which exhibits a strong phenotype. The differences in the amplitude of local mobility were more pronounced at the two end segments of the N-domain than in the central region. The M-domain is partially exposed and exhibits a higher amplitude of local mobility, indicating a lower degree of chain packing in the amyloid state, as well as a higher mobility in the Sc4 state compared to the Sc37 state. The altered local conformational dynamics in these two distinct amyloid states provide molecular insights into the varied fragility and severing efficiency that govern the inheritance patterns of strong and weak prion strains.

The Role of Structural Dynamics in Determining the Prion Strain Diversity

Prion proteins exhibit alternate structural states and are associated with a number of devastating transmissible diseases. Recent discoveries have revealed the emerging functional roles of prions in a wide range of organisms. For instance, the self-perpetuating conformational change coupled with amyloid formation of a yeast prion protein, Sup35p, a translational termination factor in yeast, is responsible for novel [PSI+] prion phenotypes in Saccharomyces cerevisiae. The 253-residue NM domain of Sup35p is an intrinsically disordered segment and is sufficient for [PSI+] prion initiation and propagation. The NM amyloid recapitulates one of the most spectacular phenomena of prions, namely, the strain-diversity. Earlier it has been shown that two well-defined strains of [PSI+] can be created in vitro. The molecular origin of these strains is postulated to involve diverse, yet related, conformational states and supramolecular packing of proteins within the amyloid fibrils. However, the precise structural and dynamical variations between the prion strains and their distinct physiological impacts remain elusive. To elucidate the structural origins of the prion strains, we took advantage of the fact that NM is devoid of tryptophan and created 19 single tryptophan mutants encompassing the entire length of NM. After establishing that these mutants behave similar to wild-type, we recorded a number of steady-state and dynamic fluorescence readouts that revealed the residue-specific dynamics and supramolecular packing within the amyloids responsible for different strains. The structural differences in two prion strains provided unique molecular insight into the differential binding of Hsp104 that is known to govern the strain propagation. The strain-diversity was further elucidated using time-resolved emission spectra that provided intriguing insights into the water relaxation dynamics within the amyloid architecture. Taken together, our results provide important biophysical clues in discerning the prion strain-diversity in a residue-specific manner.