Fig. 1. Three-dimensional structure of native HypF-N from X-ray crystallography (PDB code 1GXU).²⁰ (a) Location of the aromatic residues: Trp27 and Trp81 are shown in yellow; Tyr62 in cyan; Phe22, Phe25, Phe57, Phe79 and Phe88 in red. (b) Location of the charged residues: basic and acidic side chains are shown in red and blue sticks, respectively. A basic side chain, Lys3, is missing in the figure because the structure was not solved for the first four, highly heterogeneous N-terminal residues. This figure was created using PyMOL (DeLano Scientific LLC, San Francisco, CA).

Fig. 2. Far-UV CD, intrinsic fluorescence emission and near-UV CD spectra of HypF-N. (a and b) Spectra were acquired at 25 °C in 25 mM 3,3-dimethylglutarate, 2 mM DTT, 50 mM ionic strength, pH 5.7 (dashed line); 20 mM TFA, 50 mM ionic strength, pH 1.7 (continuous line); 20 mM TFA, 7.8 M urea (dotted line). Protein concentration was 19 and 1.9 μM for CD and fluorescence measurements, respectively. (c) Near-UV CD spectra at 25 °C of 38 μM HypF-N in 50 mM acetate, 2 mM DTT, 50 mM ionic strength, pH 5.5 (dashed line); 20 mM TFA, 50 mM ionic strength, pH 1.7 (continuous line).

Fig. 3. Sensitivity of pH-denatured HypF-N to changes of solution conditions. (a) Far-UV CD spectra of 19 μM HypF-N in 20 mM TFA, 2 mM DTT, 30 mM NaCl (50 mM ionic strength), pH 1.7 at the following temperature values: 11 °C (black); 25 °C (blue); 38 °C (green); 54 °C (red) and 73 °C (yellow). (b) Far-UV CD spectra of 19 μM HypF-N in 20 mM TFA, pH 1.7 at different ionic strength values: 20 mM (black), 50 mM (blue), 70 mM (green), 80 mM (red) and 100 mM (yellow). (c) Ratio between the total area under the fluorescence emission spectra of ANS in the presence (F) and absence (F₀) of HypF-N as a function of the ionic strength of the solution. (d) Wavelength of maximum fluorescence emission of ANS (λ_max) as a function of the ionic strength of the solution. Error bars represent standard deviation values.

Fig. 4. (a) Limited proteolysis at 25 °C of HypF-N at pH 7.0 (left), at pH 1.7 in 30 mM NaCl (middle) and at pH 1.7 in 80 mM NaCl (right) followed by RP-HPLC. The figure reports the chromatograms corresponding to 10-min reactions of HypF-N by proteinase K (pK), chimotrypsin (Ch), thermolysin (Th), pepsin (p), protease XIII from A. saitoi (p13) and protease XVIII from Rhizhopus species (p18). All the profiles are normalized to the highest peak. The identity of the protein fragments was assessed by ESI-MS (Table 1 in Supplementary Material) and is reported as labels on each RP-HPLC peak. (b) Schematic representation of the cleavage sites on HypF-N sequence by proteinase K (green) at pH 7.0 (row I) and by pepsin (light blue), protease XVIII (red) and protease XIII (yellow) at pH 1.7 in the presence of either 30 mM (row III) or 80 mM NaCl (row IV). Predicted cleavage sites for proteinase K (row II) or pepsin (row V) are also reported as well as the position of the secondary-structure elements of native HypF-N.

Fig. 5. Compactness of HypF-N under different conditions. (a) Size distributions obtained by DLS measurements performed in 25 mM sodium phosphate, 50 mM ionic strength, 2 mM DTT, pH 7.0. (b) Size distributions obtained in 20 mM TFA, 50 mM ionic strength, pH 1.7. (c) Size distributions obtained in 5 M urea in 25 mM sodium phosphate, 5.7 mM NaCl, 2 mM DTT, pH 7.0. Protein concentration was 38 μM. Each histogram reports the mean values obtained by averaging over six consecutive measurements performed at 25 °C. Error bars represent the standard deviations of these values.

Fig. 6. Aggregation of HypF-N at acidic pH and different values of ionic strength. (a and b) Time courses of the ratio between the ThT fluorescence emission values measured in the presence (F) and absence (F₀) of HypF-N. Conditions were 48 μM HypF-N, 20 mM TFA, pH 1.7, 25 °C, in the presence of different aliquots of NaCl to achieve ionic strength values of 20 mM (yellow), 50 mM (red), 80 mM (violet), 110 mM (light blue), 150 mM (blue), 200 mM (light green), 250 mM (dark green), 300 mM (brown) and 350 mM (black). Data are reported over a time scale of 24 h (a) and 800 h (b). (c and d) TM-AFM images (height data) obtained by observing a 48 μM HypF-N sample in 20 mM TFA, pH 1.7, at 80 mM ionic strength after incubation at 25 °C for 10 days (c) or 30 days (d). (e) TM-AFM images (height data) of a 48 μM HypF-N sample after 9 days of incubation in 20 mM TFA, pH 1.7, 25 °C at 350 mM ionic strength. Scan size: 1.0 μm. Z range: 5 nm (c) and 8 nm (d, e).

Fig. 7. Classification of protein conformations on the basis of [θ]₂₂₂ and [θ]₂₀₀ values. (a) [θ]₂₂₂ versus [θ]_200 nm of acid-unfolded HypF-N at low ionic strength (orange circle) and of the native state (brown circle) are reported and compared to a previously shown classification of natively unfolded proteins adopting an RC-like conformation (yellow), natively unfolded proteins adopting a PMG-like state (cyan), globular proteins adopting MG states (blue) and native states (green). The conformations of 11 globular and natively unfolded proteins under conditions in which they form amyloid fibrils are also shown (red). Adapted with permission from Ref. 10. (b) The acid-unfolded states of HypF-N populated under different conditions: no NaCl, 25 °C (pink); 30 mM NaCl, 11 °C (purple); 30 mM NaCl, 25 °C (orange); 30 mM NaCl, 73 °C (black); 80 mM NaCl, 25 °C (green). Yellow and cyan areas define the typical values for RC-like and PMG-like states, respectively.¹⁰