Protein amyloids develop an intrinsic fluorescence signature during aggregation

Abstract

We report observations of an intrinsic fluorescence in the visible range, which develops during the aggregation of a range of polypeptides, including the disease-related human peptides amyloid-β(1-40) and (1-42), lysozyme and tau. Characteristic fluorescence properties such as the emission lifetime and spectra were determined experimentally. This intrinsic fluorescence is independent of the presence of aromatic side-chain residues within the polypeptide structure. Rather, it appears to result from electronic levels that become available when the polypeptide chain folds into a cross-β sheet scaffold similar to what has been reported to take place in crystals. We use these findings to quantify protein aggregation in vitro by fluorescence imaging in a label-free manner.

Environment-transformable sequence–structure relationship: a general mechanism for proteotoxicity

“…Recently, protein fibrils have been found to absorb light in the near-UV range and to emit a structure-specific intrinsic fluorescence in the visible range even in the absence of aromatic amino acids. Most strikingly, this intrinsic visible fluorescence has been characterized to originate from the hydrogen-bonding network of protein fibrils with cross-β structures (Chan et al 2013;Lim et al 2016a;Lu et al 2016;Pinotsi et al 2016). Based on previous studies by us and other groups, a diagnostic probe of this novel intrinsic visible fluorescence can be established which is able to distinguish two different types of the fibril structures.…”

“…On the other hand, under some conditions, like the yeast prion proteins (Shorter and Lindquist 2005;Michelitsch and Weissman 2000;Chien and Weissman 2001), the TDP-43 and FUS prion-like domains have been biophysically characterized to form fibril/hydrogel structures with cross-β structures (Han et al 2012;Lim et al 2016a;Lu et al 2016;Kato and McKnight 2017;Murray et al 2017). Very unexpectedly, we discovered that the self-assembly of both TDP-43 and FUS prion-like domains into the fibril structures is highly pHdependent (Lim et al 2016a;Lu et al 2016): at low pH such as 4.0, they remained mostly monomeric for many weeks, while at neutral pH they showed a strong capacity to selfassemble into fibrils with cross-β structures as reflected by the development of intrinsic visible fluorescence, which is a novel protein fluorescence originating from the hydrogenbonding network in β-rich secondary structures (Shukla et al 2004;Chan et al 2013;Lim et al 2016a;Lu et al 2016;Pinotsi et al 2016). Furthermore, as indicated by low NMR temperature coefficients of most backbone amides, we found that, despite being intrinsically disordered, the TDP-43 prion-like domain contains a large number of intra-molecular hydrogen bonds between side chains of Ser, Thr, Asn, Gln and backbone atoms (I of Fig.…”

“…While the formation of the dynamic fibrils such as by the FUS and TDP-43 prion-like domains enriched with polar and uncharged residues (Lim et al 2016a;Lu et al 2016) is characterized by the development of the intrinsic visible fluorescence with the emission maximum at~445 nm (II of Fig. 2c), the formation of the classic amyloid fibrils by Aβ42 (Chan et al 2013) and by the hydrophobic region of the TDP-43 prion-like domain (Lim et al 2016a) is indicated by the development of the intrinsic visible fluorescence with the emission maximum at~475 nm (II of Fig. 2d).…”

“…Noticeably, the emission maximum of the intrinsic visible fluorescence of the classic amyloid fibrils (~475 nm) is significantly red-shifted as compared to that of the dynamic fibrils formed by the prion-like domains (~445 nm). This is likely due to the significant difference in the amount and arrangement of the hydrogen bond networks involved in the backbone peptide bonds of two types of fibril structures: the higher the amount and the tighter of hydrogen bonds arranged in the cross-β structures, the larger the red-shift (Chan et al 2013;Lim et al 2016a;Lu et al 2016;Song 2017;Pinotsi et al 2016). …”

Nanoscale imaging of neurotoxic proteins

“…An intriguing phenomenon recently discovered by us is that the formation of amyloid fibrils is accompanied by the adoption of a structure specific intrinsic fluorescence in the visible range, independent of the presence of aromatic residues in the polypeptide chain 10 , 11 . This provides for a new tool in the diagnostics of aggregation kinetics in vitro and has been used as a quantitative assay, for example, to study the elongation reactions of α-synuclein 12 , see Figure 2.…”