Citrate-stabilized gold nanoparticles hinder fibrillogenesis of a pathological variant of β₂-microglobulin

Abstract

Nanoparticles have repeatedly been shown to enhance fibril formation when assayed with amyloidogenic proteins. Recently, however, evidence casting some doubt about the generality of this conclusion started to emerge. Therefore, to investigate further the influence of nanoparticles on the fibrillation process, we used a naturally occurring variant of the paradigmatic amyloidogenic protein β-microglobulin (β2m), namely D76N β2m where asparagine replaces aspartate at position 76. This variant is responsible for aggressive systemic amyloidosis. After characterizing the interaction of the variant with citrate-stabilized gold nanoparticles (Cit-AuNPs) by NMR and modeling, we analyzed the fibril formation by three different methods: thioflavin T fluorescence, native agarose gel electrophoresis and transmission electron microscopy. The NMR evidence indicated a fast-exchange interaction involving preferentially specific regions of the protein that proved, by subsequent modeling, to be consistent with a dimeric adduct interacting with Cit-AuNPs. The fibril detection assays showed that AuNPs are able to hamper D76N β2m fibrillogenesis through an effective interaction that competes with protofibril formation or recruitment. These findings open promising perspectives for the optimization of the nanoparticle surface to design tunable interactions with proteins.

Short-Chain Alkanethiol Coating for Small-Size Gold Nanoparticles Supporting Protein Stability

“…The fluorescence of W60G variant is, indeed, approximately 20% lower than the value of wild-type protein. This result is consistent with a strong localization of the electrostatic interaction, probably being due to the compactness of the NP electron plasma [14,15]. The character of the interaction surface, as described in Figure 6, can be appreciated when compared to the β2m regions that are involved in the contacts with the heavy chain of type I major histocompatibility complex (MHC-I) [19].…”

“…The interaction of β2m and its variants with AuNPs has been thoroughly investigated by our group [14,15]. We found that the actual interaction between the citrate-coated surface of AuNPs and β2m is essentially of electrostatic nature, although the overall protein charge should be around zero or slightly negative.…”

“…In both of the cases, a labile interaction was observed that does not affect the overall folding and is mainly located at the N-terminal apical part of the protein structure. Furthermore, it was shown that Cit-AuNPs are able to partially hinder the fibrillogenesis of the most amyloidogenic variant, namely D76N β2m [15].…”

“…This adjustment leads to 9-12 tetramers per NP that means 36-48 monomers. If the ratio between the NP surface area and the cylindroid base is considered, then the number of protein monomers per NP is reduced to [14][15][16][17][18][19][20][21][22]. From all of these considerations, beyond any critical evaluation, the number of β2m molecules that can be adsorbed on a MPA-AuNP goes from 14 to 50, approximately.…”

“…In patients that are affected by chronic renal failure, it accumulates in the blood and precipitates into amyloid deposits in correspondence of the joints. In literature, the interaction between β2m and citrate-stabilized AuNPs (Cit-AuNPs) was reported for both the wild-type and D76N variants [14,15]. In both of the cases, a labile interaction was observed that does not affect the overall folding and is mainly located at the N-terminal apical part of the protein structure.…”