Mutation V209I is associated with familial CJD and this residue is tightly embedded in the hydrophobic core and might affect both subdomain separation as well as their reannealing. On the other hand, mutations in H2 with increased hydrophobicity, V175I and T187I, should stabilize subdomain H2–H3, and might thus facilitate separation of subdomains and conversion. Substitution of valine for isoleucine at position 209 in mPrP did not affect its stability, consistent with findings of a previous study. The effect of this mutation was proposed to lead to increased stability of the folding intermediate. Molecular dynamics simulations proposed that isoleucine might cause steric crowding in the hydrophobic core which may cause misfolding, however we observed indistinguishable in vitro conversion. Surprisingly, the mPrP V209I mutant was inefficiently converted to PrPres in HpL3- 4 cells exposed to mouse prion strain 22L. Direct prion titer analysis of different cell populations could not be performed due to expected transmission barriers introduced by PrP amino acid substitutions. However, the fact that PrPres was present in all cell lines several passages post infection argues that all PrP mutants were capable of supporting prion infection, albeit likely to different degrees. PrP mutation V210I is associated with genetic prion disease in humans, arguing that this mutation per se is not refractory to prion formation. A likely explanation is that prion strains differ in their capacities to refold a given mutant PrPC into its infectious isoform. Furthermore, differences in the human and murine PrP amino acid sequence PB 203580 supply context can influence the conversion efficiency of PrPC, as has been shown for other PrP mutants. The fact that a pathogenic PrP amino acid substitution does not generally support efficient PrPres formation once again demonstrates different degrees of compatibility of PrP mutants with different PrPSc conformers. Previously identified mutations which did not support propagation of 22L prions were mapped to the surface and were proposed to interfere with packing of PrPSc aggregates, whereas V209I lies in the hydrophobic core. Previously observed differences in the population of the folding intermediate may be the reason that conversion of this mutant under the physiological conditions proceeds slowly or towards the nonfibrillar aggregates, while in vitro strongly unfolding conditions decrease this difference. Two hydrophobic PrP mutants V175I and T187I displayed increased thermal stability in comparison to the wild-type protein, indicating that introduced isoleucines interact favorably with H3 and extend the hydrophobic core. Substitution of valine for isoleucine at position 175 of mPrP did not appreciably alter in vitro fibrillization kinetics. No overt differences in structure between wild-type and V175I PrP fibrils were observed by atomic force microscopy. Furthermore, PrP mutant V175I efficiently supported PrPres formation upon 22L prion infection when expressed in HpL3-4 cells.