title: Cystatin forms a tetramer through structural rearrangement of domain-swapped dimers prior to amyloidogenesis.
abstract: The cystatins were the first amyloidogenic proteins to be shown to oligomerize through a 3D domain swapping mechanism. Here we show that, under conditions leading to the formation of amyloid deposits, the domain-swapped dimer of chicken cystatin further oligomerizes to a tetramer, prior to fibrillization. The tetramer has a very similar circular dichroism and fluorescence signature to the folded monomer and dimer structures, but exhibits some loss of dispersion in the 1H-NMR spectrum. 8-Anilino-1-naphthalene sulfonate fluorescence enhancement indicates an increase in the degree of disorder. While the dimerization reaction is bimolecular and most likely limited by the availability of a predominantly unfolded form of the monomer, the tetramerization reaction is first-order. The tetramer is formed slowly (t(1/2)=six days at 85 degrees C), dimeric cystatin is the precursor to tetramer formation, and thus the rate is limited by structural rearrangement within the dimer. Some higher-order oligomerization events parallel tetramer formation while others follow from the tetrameric form. Thus, the tetramer is a transient intermediate within the pathway of large-scale oligomerization.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Sanders
authors.forename: C
authors.initials: C
authors.lastname: Jeremy Craven
authors.forename: Lee D
authors.initials: LD
authors.lastname: Higgins
authors.forename: Silva
authors.initials: S
authors.lastname: Giannini
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Conroy
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hounslow
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
authors.forename: Rosemary A
authors.initials: RA
authors.lastname: Staniforth
mesh_terms: D000682:Amyloid / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D015891:Cystatins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D008970:Molecular Weight; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000682:Amyloid; D015891:Cystatins; D011994:Recombinant Proteins