Cystatin forms a tetramer through structural rearrangement of domain-swapped dimers prior to amyloidogenesis |
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Authors: | Sanders Anna Jeremy Craven C Higgins Lee D Giannini Silva Conroy Matthew J Hounslow Andrea M Waltho Jonathan P Staniforth Rosemary A |
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Affiliation: | Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK. |
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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. |
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Keywords: | cystatin amyloid intermediate CAA domain-swapping dimer |
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