Fig 1.
The structural backbone and surface charge of PAF and PAFD19S.
(A) Amino acid sequence of mature PAF and PAFD19S showing the β-strands (red arrows) and the site of amino acid exchange. (B) Backbone of the structural ensemble of PAF (left) and PAFD19S (right). Arrows indicate the β-strands that are connected by loops, the Asp/Ser19 exchange is highlighted with red "sticks", respectively. (C) Surface representation of PAF (left) and PAFD19S (right) coloured according to electrostatic potential calculated in vacuum (blue: electropositive; red: electronegative). The position of amino acid exchange is indicated by an arrow.
Fig 2.
Comparison of NH-water exchange rates measured in CLEANEX NMR experiments.
The NH-water exchange rates (s-1) are shown for PAF (green) and PAFD19S (blue) at 298 K and pH = 6.0. Note that the very slow exchange rates from residues located in β-strands are not available by this technique, while measurement of fast exchange is inaccurate as shown by the error bars. Some Asn side-chain carboxamides are separately shown (to the right from the dashed line from residue 55: N18, N33, N40, N49, N50).
Fig 3.
Modelling of thermal unfolding of PAF and PAFD19S.
(A-C) Fitting of a representative residue Ala20 by the two-state (A for PAF, C for PAFD19S), and the three-state unfolding model (B) for PAF. Both folded (red ○) and unfolded (blue □) fractions are shown on A and C. While Ala20 could not be fit below 6% fit error (A, 10.01%) in case of PAF, fitting by the three-state model was successfully applied (B, fit error = 1.96%, folded: green ●, unfolded 1: orange ●, unfolded 2: blue □, sum of unfolded 1 and 2: red ○). In case of PAFD19S Ala20 is rather a two-state folder (C, fit error = 3.37%). The estimated relative error of the experimental 2D peak volumes in the 15N-1H HSQC spectra must be within 5%, according to repeated experiments. (D) Ribbon representation of PAF (left) and PAFD19S (right), displaying the error limits of the thermal unfolding experiment fits. Red: residues that could be fit by the two-state model below 6% error limit. Green: residues that could not be fit below the error limit by the two-state model. Grey: not detected residues (signals could not be integrated because of overlaps). "Sticks" represent the site of the mutation (Asp/Ser19).
Table 1.
The effect of ion supplementation of the culture medium on the growth-inhibitory activity of PAF and PAFD19S in N. crassa.
Fig 4.
The localization of BODIPY-labelled PAF and PAFD19S in 6 h-old N. crassa germlings.
After the treatment for 1 h with 32 μM BODIPY-PAF the conidial cell wall, vacuoles and the cytoplasm were stained. PAF treated hyphae showed large vacuoles (asterisks, left panels). Only weak signals at the outer layers and in the cells could be observed with BODIPY-PAFD19S. Scale bar = 20 μm.
Fig 5.
The effect of the addition of ions to the growth medium on binding and uptake of BODIPY-labelled PAF and PAFD19S.
Conidia of N. crassa were germinated in the presence of 4 μM BODIPY-PAF or BODIPY-PAFD19S for 6 h in liquid medium supplemented with 1 mM NaCl, MgCl2 and CaCl2, respectively. In the control the 0.2 x Vogel’s growth medium was without ion supplementation. Scale bar = 30 μm.
Fig 6.
The effect of PAF and PAFD19S on the [Ca2+]c resting level of 6 h-old N. crassa-AEQ germlings.
Proteins were added (arrow at time point 0) at a final concentration of 3.2 and 32 μM to the growth medium (A, C) and in medium pre-treated with 5 mM BAPTA (B, D). Top panels for PAF and bottom panels for PAFD19S. Measurements were taken every 40 sec over a period of 60 min. Samples treated with medium without the antifungal protein served as controls. Values represent the mean of three samples ± standard errors.
Fig 7.
Viability of N. crassa conidia exposed to 32 μM PAF and PAFD19S.
(A) Colony forming units were determined by counting the colonies emerging on agar plates after conidia had been treated for 1, 4 and 24 h, respectively, plated in appropriate dilutions and incubated for 24 h (untreated controls) or 48 h. Values are given in %-survival (untreated controls were set to be 100%) and represent the mean of three experiments ± standard errors. (B) Untreated conidia and conidia exposed to ethanol served as negative and positive staining controls, respectively. Conidia exposed to BODIPY-PAF (C) and BODIPY-PAFD19S (D) for 1, 4 and 24 h were co-stained with PI. Scale bar = 30 μm.