NR AXTT
AU Nyström,S.; Hammarström,P.
TI How Does Amino Acid Replacement in Position 129 of the Human Prion Protein Influence PrP Aggregation and Fibril Formation?
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.01
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: The clinical role of the polymorphism M129V in the human PrP gene is well documented. MV129 heterozygous individuals appear to be protected from prion infection. Also most cases of sporadic CJD afflict individuals homozygous either for MM or VV. Differences in codon 129 genotype give rise to differences in phenotype regarding plaque distribution in the CNS as well as clinical symptoms. Despite this clinical knowledge, little is known about the molecular background to this phenomenon.
Aims: In this study we wanted to elucidate the molecular mechanism of PrP misfolding in vitro by investigating aggregation, fibrillation kinetics and seeding propensity of different 129-mutants to discover differences on the molecular level. The variants used in this initial study were M129A, M129V, M129L, M129M, M129W and M129P. Mutants were chosen to vary hydrophobicity, stereochemistry and secondary structure preference.
Methologies: After recombinant expression and purification of recombinant human PrP90-231 mutants, aggregation was initiated by subjecting the protein to agitation under physiological conditions regarding pH, temperature and buffer salt. PrP aggregation kinetics was followed by measuring sample turbidity and the kinetics of conversion into amyloid-like fibrils was followed by Thioflavin T (ThT) fluorescence.
Results and Conclusion: Under the conditions explored so far all mutants aggregated within minutes followed by conversion into amyloid-like fibrils within a few hours as determined by ThT fluorescence. The amyloid-like conversion followed stochastic nucleation for the mutants as revealed by variations of the lag-phase from chemically identical samples. Hydrophobicity at position 129 (M129, M129V, M129L, M129A, M129W) did not appear to be rate determining for the fibril conversion whereas the M129P mutant appeared to show faster conversion into ThT positive fibrils.
AD S. Nystrom, Per Hammarström, Linköping University, IFM-Chemistry, Sweden
SP englisch
PO Schottland
EA pdf-Datei und Poster (abweichender Titel: How Does Amino Acid Replacement in Position 129 of the Human Prion Protein Influence Fibril Formation?)