Excitation Energy Migration Unveils Fuzzy Interfaces within the Amyloid Architecture

Abstract

Amyloid fibrils are highly ordered nanoscopic protein aggregates comprising a cross-b amyloid core and are associated with deadly human diseases. Structural studies have revealed the supramolecular architecture of a variety of diseaseassociated amyloids. However, the critical role of transient intermolecular interactions between the disordered polypeptide segments of protofilaments in directing the supramolecular structure and nanoscale morphology remains elusive. Here, we present a unique case to demonstrate that interchain excitation energy migration via intermolecular homo-Fö rster resonance energy transfer can decipher the architecture of amyloid fibrils of human a-synuclein. Site-specific homo-Fö rster resonance energy transfer efficiencies measured by fluorescence depolarization allowed us to construct a two-dimensional proximity correlation map that defines the supramolecular packing of a-synuclein within the fibrils. These studies captured unique heteroterminal cross talks between the fuzzy interprotofilament interfaces of the parallel-in-register amyloid spines. Our results will find applications in discerning the broader role of protein disorder and fuzziness in steering the distinct polymorphic amyloids that exhibit strain-specific disease phenotypes.

Spatiotemporal modulations in heterotypic condensates of prion and α-synuclein control phase transitions and amyloid conversion

“…PrP 23–144 (Y145Stop), PrP 112–231, and single cysteine variants of full-length PrP (W31C, W99C, and S230C) were created using site-directed mutagenesis 60 , 88 . Recombinant full-length human α-Syn (1–140) plasmid cloned in vector pT7.7 was transformed in BL21(DE3)pLysS 89 . α-Syn (1–102) (N103Stop) and α-Syn (1–132) (Y133Stop) were created using the full-length α-Syn plasmid.…”

“…The primers used for α-Syn mutations are listed in Table S1 . Single cysteine variants of α-Syn (A18C, A90C, and A124C) were created using site-directed mutagenesis 89 . Recombinant prion protein constructs were overexpressed and purified using nickel-NTA affinity chromatography 60 , 88 .…”

Spatiotemporal Modulations in Heterotypic Condensates of Prion and α-Synuclein Control Phase Transitions and Amyloid Conversion

“…63,90 Recombinant full-length human α-Syn (1-140) plasmid cloned in vector pT7.7 was transformed in BL21(DE3)pLysS. 91 α-Syn (1-102) (N103Stop) and α-Syn (1-132) (Y133Stop) were created using the full-length α-Syn plasmid. The primers used for α-Syn mutations are listed in Table S1.…”

“…Single cysteine variants of α-Syn (A18C, A90C, and A124C) were used as described previously. 91 Recombinant prion protein constructs were overexpressed and purified using previously published protocols. 63,90 The purified proteins were refolded using the PD10 column in 14 mM MES buffer, pH 6.8.…”

“…Recombinant α-Syn constructs except 103Stop were purified using the anion-exchange chromatography as described previously. 91 α-Syn 103Stop was purified using cation-exchange chromatography. The purified proteins were dialyzed overnight for buffer exchange (14 mM MES buffer, pH 6.8).…”