Thioflavin T-Silent Denaturation Intermediates Support the Main-Chain-Dominated Architecture of Amyloid Fibrils

Abstract

Ultrasonication is considered one of the most effective agitations for inducing the spontaneous formation of amyloid fibrils. When we induced the ultrasonication-dependent fibrillation of β2-microglobulin and insulin monitored by amyloid-specific thioflavin T (ThT) fluorescence, both proteins showed a significant decrease in ThT fluorescence after the burst-phase increase. The decrease in ThT fluorescence was accelerated when the ultrasonic power was stronger, suggesting that this decrease was caused by the partial denaturation of preformed fibrils. The possible intermediates of denaturation retained amyloid-like morphologies, secondary structures, and seeding potentials. Similar denaturation intermediates were also observed when fibrils were denatured by guanidine hydrochloride or sodium dodecyl sulfate. The presence of these denaturation intermediates is consistent with the main-chain-dominated architecture of amyloid fibrils. Moreover, in the three types of denaturation experiments conducted, insulin fibrils were more stable than β2-microglobulin fibrils, suggesting that the relative stability of various fibrils is independent of the method of denaturation.

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

“…It appears, then, that the DLPC-induced changes in ThT fluorescence and morphological differences observed by TEM are the result of more surface level disruptions. It has previously been shown that A␤ fibrils can adopt a pseudotransition state, termed the molten globule state, in which side chain packing at the fiber surface becomes perturbed (22). The molten globule state was defined by a reduction in ThT fluorescence and truncation of the fibrillar structure while the ␤-strand signal was maintained in CD spectroscopy, similar to what we have observed in the presence of DLPC (28).…”

“…It is likely that DLPC is sufficiently mild in its perturbations so as to not fully denature and instead stabilize this unfolding transition state. Although we observed that DLPC inhibits A␤ aggregation through a combination of detergent-like interactions and stabilization of membrane-associated globulomers, it is likely that the fibrils are primarily altered through interactions with the surface of DLPC LUVs, which promote disorder in the traditionally ordered side chain packing of the fibril (22,60). This increased disorder can facilitate a reduction in ThT binding and fluorescence.…”

“…Surprisingly, it was observed that DLPC bilayers are also capable of remodeling mature A␤ fibrils into a semi-unfolded intermediate, a feat previously observed only after treatment with high concentrations of denaturants or exposure to high frequency sonication (22). These results show that thin bilayers are able to dramatically alter the aggregation of A␤ compared with conventional lipid bilayers, and this redirected misfolding can stabilize distinct, membrane-associated species not observed previously.…”

“…These results confirm that DLPC LUVs are capable of remodeling mature A␤ aggregates, although not to the extent of some traditional studies of disaggregation (49 -51). There are, then, three likely mechanisms by which DLPC LUVs may remodel preformed A␤ fibrils: (i) DLPC changes the monomer-fiber equilibrium and causes additional monomers of A␤ to enter solution via dissociation from the ends of fibers, resulting in shorter aggregates (52); (ii) DLPC LUVs alter the hydrogen bonding network found within the fibril, generating distinct polymorphs of the aggregates that are less stable and can potentially become more similar to the immature protofibril structure (52); or (iii) DLPC LUVs rearrange the side chain packing of the mature fibrils and induce a partially unfolded state without significantly altering the hydrogen bonding of the fiber core (22).…”

“…This molten globule state has previously only been observed following treatment of mature aggregates with ultrasonication. Treatment of fibrils with conventional chemical denaturants, such as detergents or guanidine hydrochloride, has previously resulted in the generation of the unfolded, monomeric form of A␤ (22). It is likely that DLPC is sufficiently mild in its perturbations so as to not fully denature and instead stabilize this unfolding transition state.…”