Detection and Characterization of Aggregates, Prefibrillar Amyloidogenic Oligomers, and Protofibrils Using Fluorescence Spectroscopy

Abstract

Transthyretin (TTR) is a protein linked to a number of different amyloid diseases including senile systemic amyloidosis and familial amyloidotic polyneuropathy. The transient nature of oligomeric intermediates of misfolded TTR that later mature into fibrillar aggregates makes them hard to study, and methods to study these species are sparse. In this work we explore a novel pathway for generation of prefibrillar aggregates of TTR, which provides important insight into TTR misfolding. Prefibrillar amyloidogenic oligomers and protofibrils of misfolded TTR were generated in vitro through induction of the molten globule type A-state from acid unfolded TTR through the addition of NaCl. The aggregation process produced fairly monodisperse oligomers (300-500 kD) within 2 h that matured after 20 h into larger spherical clusters (30-50 nm in diameter) and protofibrils as shown by transmission electron microscopy. Further maturation of the aggregates showed shrinkage of the spheres as the fibrils grew in length, suggesting a conformational change of the spheres into more rigid structures. The structural and physicochemical characteristics of the aggregates were investigated using fluorescence, circular dichroism, chemical cross-linking, and transmission electron microscopy. The fluorescent dyes 1-anilinonaphthalene-8-sulfonate (ANS), 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS), 4-(dicyanovinyl)-julolidine (DCVJ), and thioflavin T (ThT) were employed in both static and kinetic assays to characterize these oligomeric and protofibrillar states using both steady-state and time-resolved fluorescence techniques. DCVJ, a molecular rotor, was employed for the first time for studies of an amyloidogenic process and is shown useful for detection of the early steps of the oligomerization process. DCVJ bound to the early prefibrillar oligomers (300-500 kD) with an apparent dissociation constant of 1.6 muM, which was slightly better than for ThT (6.8 muM). Time-resolved fluorescence anisotropy decay of ANS was shown to be a useful tool for giving further structural and kinetic information of the oligomeric aggregates. ThT dramatically increases its fluorescence quantum yield when bound to amyloid fibrils; however, the mechanism behind this property is unknown. Data from this work suggest that unbound ThT is also intrinsically quenched and functions similarly to a molecular rotor, which in combination with its environmental dependence provides a blue shift to the characteristic 482 nm wavelength when bound to amyloid fibrils.

Morphology‐Specific Inhibition of β‐Amyloid Aggregates by 17β‐Hydroxysteroid Dehydrogenase Type 10

“…[61] Using a combination of fluorescence methods and structural techniques, it has been shown that amyloid aggregation at pH 5.8 is characterized by the rapid formation (~ 10 seconds) of amorphous aggregates with sizes ranging from 50 to 500 nm. [40] Importantly, as determined by circular dichroism (CD), these aggregates do not contain -helical or -structure and they are unable to seed fibril formation. [40] It has also been demonstrated that the aggregates evolve into smaller structures (30-80 nm) after 30 minutes which agrees well with the time window of the aggregation process observed in our FSQ and ThT experiments ( Figure 5).…”

“…[40] Importantly, as determined by circular dichroism (CD), these aggregates do not contain -helical or -structure and they are unable to seed fibril formation. [40] It has also been demonstrated that the aggregates evolve into smaller structures (30-80 nm) after 30 minutes which agrees well with the time window of the aggregation process observed in our FSQ and ThT experiments ( Figure 5). The time scale of the initial growth phase is within our mixing time, so it is likely that with the time resolution of our assay, we are monitoring the disappearance of the initially formed aggregates as they slowly evolve into other morphologies.…”

“…[43] Secondly, aggregation protocols to reproducibly promote the formation of these A aggregates are well-established and the resulting aggregates have been extensively characterized using structural imaging methods including atomic-force microscopy (AFM) and electron-microscopy (EM). [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] In a previous study we have already shown using EM that the presence of the HiLyte Fluor 555 dye at the N-terminus of the A(1-42) peptide (A 555 ) does not affect the aggregation kinetics or the morphology of the aggregates. [34] Indeed, we observed amyloid structures of A 555 with diameters of ~ 4 nm (fibrils) and ~ 22 nm (globules) which are identical to those obtained in the absence of N-terminal functionalization.…”

“…Although amyloid staining methods based on extrinsic probes, such as ThT, are known to suffer from a decrease in affinity as high as 30-fold when the pH decreases from pH 8.5 to pH 6, [40] Figure 5B, C). The observation using ThT that 17-HSD10 does not inhibit aggregation at pH 6 is therefore entirely consistent with our analysis using FSQ.…”

C-terminal sequence of amyloid-resistant type F apolipoprotein A-II inhibits amyloid fibril formation of apolipoprotein A-II in mice

“…We present a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro and believe that this system will be useful for the development of novel therapies. amyloid fibril formation | apolipoprotein A-II | inhibitory peptide | mouse | senile amyloidosis A myloidosis refers to a group of protein structural disorders characterized by the extracellular deposits of insoluble amyloid fibrils resulting from abnormal conformational changes (1)(2)(3)(4)(5). Amyloid fibrils have a characteristic ultrastructural appearance and a β-pleated sheet core structure that consists of full-length proteins and/or fragments of either WT or mutant proteins found in familial diseases (2,(6)(7)(8).…”