Proton Transfer and Structure-Specific Fluorescence in Hydrogen Bond-Rich Protein Structures

Abstract

Protein structures which form fibrils have recently been shown to absorb light at energies in the near UV range and to exhibit a structure-specific fluorescence in the visible range even in the absence of aromatic amino acids. However, the molecular origin of this phenomenon has so far remained elusive. Here, we combine ab initio molecular dynamics simulations and fluorescence spectroscopy to demonstrate that these intrinsically fluorescent protein fibrils are permissive to proton transfer across hydrogen bonds which can lower electron excitation energies and thereby decrease the likelihood of energy dissipation associated with conventional hydrogen bonds. The importance of proton transfer on the intrinsic fluorescence observed in protein fibrils is signified by large reductions in the fluorescence intensity upon either fully protonating, or deprotonating, the fibrils at pH = 0 or 14, respectively. Thus, our results point to the existence of a structure-specific fluorophore that does not require the presence of aromatic residues or multiple bond conjugation that characterize conventional fluorescent systems. The phenomenon may have a wide range of implications in biological systems and in the design of self-assembled functional materials.

Carbonyl-based blue autofluorescence of proteins and amino acids

“…The emission of dbAF from amyloid fibrils and protein aggregates has been associated with the formation of intermolecular hydrogen bonds as source of electron delocalization. [7, 9, 19] The presence of dbAF from monomeric proteins and their intramolecular hydrogen bonds would be consistent with this interpretation. However, we wanted to explore how far below the structure of folded proteins one could go while preserving dbAF emission.…”

Fluorescence Self-Quenching from Reporter Dyes Informs on the Structural Properties of Amyloid Clusters Formed in Vitro and in Cells

“…7−12 A structural characterization of amyloid clusters is thus an important topic of research in the field. Various approaches have been employed to reveal the morphology of individual amyloid fibrils or clusters directly, for instance, electron microscopy (EM), 2,5,8,12,13 atomic-force microscopy (AFM), 11,12,14−16 and super-resolution fluorescence microscopy. 17−22 However, such methods are slow to perform and can require elaborate sample preparation protocols, making them impractical to perform for screening applications or the analysis of large data sets.…”

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.…”

“…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). …”