title: An alternative structural isoform in amyloid-like aggregates formed from thermally denatured human γD-crystallin.
abstract: The eye lens protein γD-crystallin contributes to cataract formation in the lens. In vitro experiments show that γD-crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV-B photodamage results in its C-terminal domain forming the β-sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet-like aggregates that contain cross-linked oligomers of the protein, according to transmission electron microscopy and SDS-PAGE. We use two-dimensional infrared spectroscopy to show that these aggregates have an amyloid-like secondary structure with extended β-sheets, and use isotope dilution experiments to show that each protein contributes approximately one β-strand to each β-sheet in the aggregates. Using segmental (13) C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N-terminal and C-terminal domains contribute to β-sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N-terminal domain into the fiber structure to intermolecular cross linking.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706.
authors.forename: Sean D
authors.initials: SD
authors.lastname: Moran
authors.forename: Tianqi O
authors.initials: TO
authors.lastname: Zhang
authors.forename: Martin T
authors.initials: MT
authors.lastname: Zanni
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000528:radiation effects; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000737:chemistry / Q000528:radiation effects; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D038222:gamma-Crystallins / Q000737:chemistry* / Q000528:radiation effects*
chemical_list: D000682:Amyloid; C504903:CRYGD protein, human; D020033:Protein Isoforms; D038222:gamma-Crystallins
keywords: 2D IR spectroscopy; aggregate; amyloid; cataract; cross-linking; crystallin; infrared; isotope labeling