NR AWNW
AU Sjolander,D.; Nilsson,P.K.P.; Almstedt,K.; Herland,A.; Inganas,O.; Stabo-Eeg,F.; Lindgren,M.; Hammarström,P.
TI Misfolding and aggregation of caprion - a prion protein and carbonic anhydrase chimaera
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions S-22
PT Konferenz-Poster
AB There are numerous conformational diseases, such as Alzheimer's disease and the prion diseases, which are caused by aberrant protein folding. The misfolding process often originates from partly folded/unfolded protein. This state resembles the molten-globule state (MG) that is observed for many proteins, among them human carbonic anhydrase II, (HCA II). These intermediates are extensively populated at physiological conditions for heritable mutants of HCA II associated with marble brain disease. The MG of HCA II is known to aggregate by specific interactions of the central part of the ß-sheet. This central part, ß-strands 3-7, of HCA II contains a vast hydrophobic core. In this study we show that a peptide corresponding to the sequence ß-strand 6 (DGLAVLGI) forms microcrystalline flake-like assemblies resembling reported crystals of a seven residue peptide of the Sup35 yeast prion. Identical crystals were formed from an alanine-glycine rich palindrome sequence of the human prion protein, HuPrP113-120 (AGAAAAGA) despite that this sequence is predicted to adopt a helical structure. Hence this portion of HuPrP is a polymorphic stretch possibly involved in helix-sheet conversion during prion amyloid formation. We created a chimera protein, designated CAPRION, of HCA II by replacing ß-strand 6 with the HuPrP113-120 sequence. The CAPRION protein was found to exist in a MG-like state under physiological conditions as assessed by ANS binding and tryptophan fluorescence. Unfolding by the chemical denaturants GdmCl and urea reveals a highly destabilized structure. The aggregation proprieties of CAPRION compared to HCA II with and without presence of seeds of the HuPrPP113-120-peptide, was assessed by measuring the light scattering of formed aggregates in a pH-dependent aggregation assay. The results show that seeding with the peptide microcrystals induced different conformations of the aggregates. Thus, it is likely that the seeds of microcrystals of the HuPrP113-120-peptide can template aggregation by sequence independent interactions during aggregation. The aggregates were characterized with epi-fluorescence microscopy after staining with the dyes Nile Red, Congo Red, PTAA and POMT. The morphology of the aggregates has been classified into two major classes, asymmetrical aggregates and metacrystalline lamellar aggregates. The two types of aggregates are present at the same time; the latter being more abundant when seeding.
AD D. Sjolander, P.K.P. Nilsson, K. Almstedt, A. Herland, O. Inganas, Per Hammarström: IFM - Department of Physics, Chemistry and Biology, University of Linkoping, SE-581 83 Linkoping, Sweden; F. Stabo-Eeg, M. Lindgren: Department of physics, Norwegian University of Science and Technology, 7491 Trondheim, Norway. E-mail: perha@ifm.liu.se
SP englisch
PO Italien