NR ATFN
AU Rezaei,H.; Eghiaian,F.; Daubenfeld,T.; Quenet,Y.; Choiset,Y.; Haertle,T.; Grosclaude,J.
TI Conformational dynamics and oligomerization pathways of prion protein investigated by rational mutagenesis
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Oral sessions ORAL-18
PT Konferenz-Vortrag
AB
In order to obtain a deeper insight into the molecular mechanisms of PrPc/PrPsc conversion, we studied the kinetic pathway of heat induced oligomerization of the full length recombinant ovine PrP (ARQ) at pH 4.0. According to size exclusion chromatography experiments, three sets of beta enriched oligomers were generated from partial unfolding of the monomer. The oligomerization kinetics as a function of initial monomer concentration is in favor of at least three parallel oligomerization pathways rather than one sequential process.
In order to determine which PrP domain is implicated in the oligomerization process, we designed three cysteine double mutants leading to different covalently attached regions of PrP: Y160C-M209C, G130C-R167C, V169C-E224C. Oligomerization studies of these mutants revealed that i) oligomerization ignition can only occur if region H1-S2 can be separated from the region H2-H3; ii) the oligomerization pathway depends strongly on the conformation of the hinge loop between S2-H2.
Based on sheep ARQ variant calorimetric studies and the oligomer distribution pattern of mouse and human PrP protein, we designed some single mutations which affect dramatically and selectively the PrP protein oligomerization process. The mutation I208M, localized at the middle of H3 in the native sate, selectively led to the formation of PrP dodecamer after heat induced partial unfolding. On the other hand, the mutation H190A selectively led to the formation of PrP 36mer.
The existence of at least three distinct oligomerization pathways and the effect of single mutations in the oligomerization process reveals the conformational diversity of the PrP protein and a possible relationship with prion strain phenomena. The identification of domains which are implicated in the conversion process may also lead to a better understanding of the effect of mutations or PrP gene polymorphisms on the evolution of prion pathology.
AD H.Rezaei, F.Eghiaian, Y.Quenet, Y.Choiset, T.Haertle, J.Grosclaude, Institut National de Recherche Agronomique (INRA), France; T.Daubenfeld, Laboratoire des Mécanismes Réactionnels, Ecole Polytechnique, 91128 Palaiseau, France
SP englisch
PO Deutschland