Self-Assembly of Ovalbumin Amyloid Pores: Effects on Membrane Permeabilization, Dipole Potential, and Bilayer Fluidity

Amyloid assembly is inherently a stochastic and a hierarchical process comprising the genesis of heterogeneous, transiently populated prefibrillar aggregates that are characterized to be non-native oligomeric conformers. These oligomers could be either off-pathway or on-pathway species en route to amyloid fibrils that are associated with a variety of neurodegenerative disorders, namely, Alzheimer’s disease, Parkinson’s disease, and prion disease, as well as in localized and systemic amyloidoses (type II diabetes and dialysis related, respectively). Morphological characterizations of these prefibrillar aggregates indicated that apparently the doughnut or annular structure is commonly shared among various prefibrillar species irrespective of the diverse native structures and aggregation mechanisms. In this work, we have elucidated the self-assembly mechanism of amyloid pore formation from ovalbumin using a range of biophysical techniques that shed light on the time-dependent protein structural changes as aggregation progressed. Additionally, on the basis of several pieces of evidence suggesting amyloid pore-mediated cytotoxicity, we have investigated the annular amyloid–membrane interaction using a comprehensive biophysical approach. The influences of annular pores on the intramembrane dipole potential and bilayer fluidity, as a consequence of membrane permeabilization, were examined in a protein concentration- and time-dependent manner that provided important insights into the pore–membrane interactions. Instantaneous membrane permeabilization kinetics suggested that plausibly a detergent-like carpet mechanism during membrane disruption was effective. Moreover, it was inferred that a loss of membrane integrity resulted in the generation of both disordered lipid and disoriented water dipoles that reside in the immediate vicinity of the membrane bilayer. These key findings may have implications in amyloid-pore-induced deleterious effects during amyloid–membrane interactions

Structural and Dynamical Insights into the Molten-Globule Form of Ovalbumin

Ovalbumin is a 45 kDa egg-white glycoprotein which belongs to the class of serpin superfamily. We have studied the structural properties of both native and partially unfolded molten-globule forms of ovalbumin using a diverse array of spectroscopic tools. Time-resolved fluorescence measurements provided important structural and dynamical insights into the native and molten-globule states. Fluorescence anisotropy decay analysis indicated that there is a conformational swelling from the native to the molten-globule form of ovalbumin. We have also carried out red-edge excitation shift measurements to probe the dipolar relaxation dynamics around the intrinsic tryptophan residues. Additionally, stopped-flow fluorescence experiments revealed that the conformational transition from the native to the molten-globule form proceeds in a stepwise manner involving a burst-phase with a submillisecond conformational change followed by biphasic slower conformational reorganizations on the millisecond time scale leading to the final molten-globule state

pH-Responsive Mechanistic Switch Regulates the Formation of Dendritic and Fibrillar Nanostructures of a Functional Amyloid

In contrast to pathological amyloids, functional amyloids are involved in crucial physiological functions. For instance, the melanosomal protein comprising a highly amyloidogenic polypeptide repeat domain assembles into amyloid fibrils that act as templates for melanin biosynthesis within acidic melanosomes. However, the mechanism–morphology–function relationship of functional amyloids is poorly understood. Here, we demonstrate that the repeat domain of the melanosomal protein exhibits two distinct types of aggregation pathways that display nanoscale polymorphism in acidic pH. In the pH range of 4.5–6, aggregation proceeds via a typical nucleation-dependent mechanism, resulting in the formation of highly ordered β-rich curvy thread-like fibrils. On the contrary, at pH < 4.5, aggregation occurs through a rapid nucleation-independent isodesmic polymerization process that yields dendritic aggregates having lower degree of internal packing. These dendritic nanostructures can be converted into more stable fibrils by switching the pH. The nanoscale polymorphism associated with the mechanistic switch is likely to be mediated by the altered conformational propensities and intermolecular interactions due to the protonation/deprotonation of critical glutamate residues. We propose that this striking shift in the mechanism that dictates the nanoscale morphology regulates the melanosomal maturation

Conformational Switching and Nanoscale Assembly of Human Prion Protein into Polymorphic Amyloids via Structurally Labile Oligomers

Conformational switching of the prion protein (PrP) from an α-helical normal cellular form (PrP^C) to an aggregation-prone and self-propagating β-rich scrapie form (PrP^Sc) underlies the molecular basis of pathogenesis in prion diseases. Anionic lipids play a critical role in the misfolding and conformational conversion of the membrane-anchored PrP into the amyloidogenic pathological form. In this work, we have used a diverse array of techniques to interrogate the early intermediates during amyloid formation from recombinant human PrP in the presence of a membrane mimetic anionic detergent such as sodium dodecyl sulfate. We have been able to detect and characterize two distinct types of interconvertible oligomers. Our results demonstrate that highly ordered large β-oligomers represent benign off-pathway intermediates that lack the ability to mature into amyloid fibrils. On the contrary, structurally labile small oligomers are capable of switching to an ordered amyloid-state that exhibits profound toxicity to mammalian cells. Our fluorescence resonance energy transfer measurements revealed that the partially disordered PrP serves as precursors to small amyloid-competent oligomers. These on-pathway oligomers are eventually sequestered into higher order supramolecular assemblies that conformationally mature into polymorphic amyloids possessing varied nanoscale morphology as evident by the atomic force microscopy imaging. The nanoscale diversity of fibril architecture is attributed to the heterogeneous ensemble of early obligatory oligomers and offers a plausible explanation for the existence of multiple prion strains <i>in vivo</i>

Nanoscopic Amyloid Pores Formed via Stepwise Protein Assembly

Protein aggregation leading to various nanoscale assemblies is under scrutiny due to its implications in a broad range of human diseases. In the present study, we have used ovalbumin, a model non-inhibitory serpin, to elucidate the molecular events involved in amyloid assembly using a diverse array of spectroscopic and imaging tools such as fluorescence, laser Raman, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images revealed a progressive morphological transition from spherical oligomers to nanoscopic annular pores that further served as templates for higher-order supramolecular assembly into larger amyloid pores. Raman spectroscopic investigations illuminated in-depth molecular details into the secondary structural changes of the protein during amyloid assembly and pore formation. Additionally, Raman measurements indicated the presence of antiparallel β-sheets in the amyloid core. Overall, our studies revealed that the protein conformational switch in the context of the oligomers triggers the hierarchical assembly into nanoscopic amyloid pores. Our results will have broad implications in the structural characterization of amyloid pores derived from a variety of disease-related proteins

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

Water Rearrangements upon Disorder-to-Order Amyloid Transition

Water plays a critical role in governing the intricate balance between chain-chain and chain-solvent interactions during protein folding, misfolding, and aggregation. Previous studies have indicated the presence of different types of water in folded (globular) proteins. In this work, using femtosecond and picosecond time-resolved fluorescence measurements, we have characterized the solvation dynamics from ultrafast to ultraslow time scale both in the monomeric state and in the amyloid state of an intrinsically disordered protein, namely κ-casein. Monomeric κ-casein adopts a compact disordered state under physiological conditions and is capable of spontaneously aggregating into highly ordered β-rich amyloid fibrils. Our results indicate that the mobility of “biological water” (type I) gets restrained as a result of conformational sequestration during amyloid formation. Additionally, a significant decrease in the bulk water component with a concomitant increase in the ultraslow component revealed the ordering of trapped interstitial water (type II) upon disorder-to-order amyloid transition. Our results provide an experimental underpinning of significant water rearrangements associated with both chain desolvation and water confinement upon amyloid formation