title: Considerably Unfolded Transthyretin Monomers Preceed and Exchange with Dynamically Structured Amyloid Protofibrils.
abstract: Despite numerous studies, a detailed description of the transthyretin (TTR) self-assembly mechanism and fibril structure in TTR amyloidoses remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry (HXMS) analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher average solvent exposure of TTR upon fibrillation. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state. Together, these data reveal that the fibrillar state interchanges with the solution state. Accordingly, we suggest that TTR fibrillation proceeds via addition of considerably unfolded monomers, and the continuous presence of amyloidogenic structures near the protofibril surface offers a plausible explanation for secondary nucleation. We argue that the presence of such dynamic structural equilibria must impact future therapeutic development strategies.
authors.affiliation: Department of Pharmacy and Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.|IFM - Department of Chemistry, Linköping University, Linköping, Sweden.
authors.forename: Minna
authors.initials: M
authors.lastname: Groenning
authors.affiliation: IFM - Department of Chemistry, Linköping University, Linköping, Sweden.
authors.forename: Raul I
authors.initials: RI
authors.lastname: Campos
authors.affiliation: IFM - Department of Chemistry, Linköping University, Linköping, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hirschberg
authors.affiliation: IFM - Department of Chemistry, Linköping University, Linköping, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Hammarström
authors.affiliation: Department of Pharmacy and Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Bente
authors.initials: B
authors.lastname: Vestergaard
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D041961:Deuterium Exchange Measurement; D013058:Mass Spectrometry; D046529:Microscopy, Electron, Transmission; D011228:Prealbumin / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D053838:Scattering, Small Angle; D017550:Spectroscopy, Fourier Transform Infrared; D014961:X-Ray Diffraction
chemical_list: D000682:Amyloid; D011228:Prealbumin