title: New insights into the self-assembly of insulin amyloid fibrils: an H-D exchange FT-IR study.
abstract: The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 +/- 2% of amide protons are protected. Of those "trapped" protons, a further 25 +/- 2 or 35 +/- 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25 degrees C or 0.1 MPa and 100 degrees C, respectively. In contrast to the native or unfolded protein, fibrils do not H-D exchange upon incubation at 65 degrees C. A complete deuteration of H(2)O-grown fibrils occurs when the beta-sheet structure is reassembled in a 75 wt % DMSO/D(2)O solution. Our findings suggest a densely packed environment around the amide protons involved in the intermolecular beta-sheet motive. In disagreement with the concept of "amyloid fibers as water-filled nanotubes" [Perutz, M. F., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5591-5595], elution of D(2)O-grown fibrils with H(2)O is complete, which is reflected by the vanishing of D(2)O bending vibrations at 1214 cm(-)(1). This implies the absence of "trapped water" within insulin fibrils. The rigid conformations of the native and fibrillar insulin contrast with transient intermediate states docking at the fibrils' ends. Room-temperature seeding is accompanied by an accelerated H-D exchange in insulin molecules in the act of docking and integrating with the seeds, proving that the profound structural disruption is the sine qua non of forming an aggregation-competent conformation.
authors.affiliation: Institute of High-Pressure Physics, Polish Academy of Sciences, Sokolowska 29/37, 01-142 Warsaw, Poland. wdzwolak@unipress.waw.pl
authors.forename: Wojciech
authors.initials: W
authors.lastname: Dzwolak
authors.forename: Anna
authors.initials: A
authors.lastname: Loksztejn
authors.forename: Vytautas
authors.initials: V
authors.lastname: Smirnovas
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D003903:Deuterium; D004121:Dimethyl Sulfoxide; D006859:Hydrogen; D007328:Insulin / Q000737:chemistry*; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013816:Thermodynamics
chemical_list: D000682:Amyloid; D007328:Insulin; D006859:Hydrogen; D003903:Deuterium; D004121:Dimethyl Sulfoxide