Figure 1.
Structure and sequence conservation of the HET-s prion forming domain.
A. The structure of the first pseudo repeat (residue 226–246) as well as the second repeat and the C-terminal loop region (residue 262–289) are given (after PDB 2KJ3). The 32 residues that have been mutated are coloured. Colour coding is as follows, polar residues in green, hydrophobic in blue, aromatic in cyan, positively charge in red, negatively charged in magenta and glycine in yellow. B. Sequence alignment of the PFD region of various HET-S homologs from various fungal species. The 21 amino acid repeats are boxed. The position of the β-strands of the β-solenoid core is represented as blue arrows C. Consensus sequence of the first and second repeats of the HET-S homologs presented in the alignment in B. The consensus was generated using MEME. In this graphical representation, the size of the letter reflects the level of conservation of the corresponding residue, the scale is given in information content measured in bits.
Figure 2.
Effect of alanine mutations on [Het-s] activity in vivo.
Δhet-s strain was transformed with the given mutants and the fraction of transformants producing a barrage reaction to [Het-S] (dark grey) and able to convert a [Het-s*] to the prion phenotype (light grey) is given in %. The mutants were grouped into three functional groups: mutants with wild-type or close to wild-type activity (>60%) were labeled by a green dot, slightly affected mutants (>30, <60%) were labeled by an orange dot, and strongly affected mutants (<30%) by a red dot, respectively. The “*” sign indicates that no transformant displayed the given activity.
Figure 3.
Propagation rate of [Het-s] in single copy integrants of alanine mutants.
A. For wt and each mutant, the rate of [Het-s] propagation was measured in the experimental setting in which the length of the barrage line gives the distance of [Het-s] spreading in a period of 10 hours. B. Mean propagation distance in at least 4 experiments with standard deviation.
Table 1.
Spontaneous [Het-s] prion formation rate for alanine mutants of HET-s.
Table 2.
Spore-killing activity of selected het-s mutants in a cross a with het-S male parent.
Figure 4.
2D Solid state NMR spectra of Ala variants of HET-s(218–289) amyloid fibrils.
The 2D DARR solid state NMR spectra of 15N,13C-labeled Ala variants of HET-S(218–289) amyloid fibrils are shown on top of the corresponding spectrum of wild-type HET-s(218–289) amyloid fibrils, the latter which contour lines are color-coded black. The spectra are labelled according to the amino acid replacement. The close resemblance between the variant and the wild-type spectra indicates the conservation of the β-solenoid fold in the variants with the exception for G278A and the double variant F286A/W287A. In Figure S3 the same spectra are shown including the assignment of wild-type HET-s(218–289) amyloid fibrils [29].
Figure 5.
GuHCl unfolding measurements of HET-s(218–289) amyloid fibrils and Ala variants thereof.
The GuHCl denaturation curve of amyloid fibrils of HET-s(218–289) and Ala variants (as indicated) were measured by the OD500 (y-axis) at various GuHCl concentration (x-axis) after incubation in the corresponding GuHCl buffers for one day. The change of the OD500 value is indicative of the loss of large aggregated protein species. Three samples each were incubated and the individual measurements are highlighted by asterisks, by small dots, or by open circles, respectively. Individual successful fits following the concept by Santoro and Bolen to study protein folding and unfolding [61] are shown by black lines and the calculated change in Gibbs energy (ΔG) with its standard deviation from the three measurements are listed in Table 3.
Table 3.
Gibbs free unfolding energy of HET-s(218–289) amyloid fibrils.
Figure 6.
Expression of a HET-s-GFP F286A and W287A fusion proteins in P. anserine.
HET-s-GFP F286A and W287A were expressed in Δhet-s P. anserina strain or the same strain over-expressing wild-type HET-s. Note that both mutants are unable to form dot-like aggregates in Δhet-s but can be incorporated in dot-like aggregates in the presence of wild-type HET-s.
Table 4.
Infectivity of recombinant HET-s(218-289) F286A and W287 fibrils.