Figure 3.
Diagonal-compensated 4D 1H/1H correlations in spectra of hydrophobin rodlets lacking crystalline order. (A) Negative-stain EM image of EASΔ15 hydrophobin rodlets. (B) Surface charge representation of the amphipathic hydrophobin monomer (PDB 2K6A). (C) Hypothetical model of the rodlet structure as a functional fungal amyloid obtained using molecular docking with the solution structure of monomeric EASΔ15 and mutagenesis and other biophysical data.76 (D) 1H amide resolution of the rodlets (black, bottom) compared to the microcrystalline SH3 domain (gray, top). Both spectra are shown without apodization or truncation of the FID. (E and F) Representative 2D slices from an HNNH 4D experiment. Diagonal compensation was achieved (black contours) applying 1 out of 5 scans without dipolar recoupling. (Gray contours display noncompensated scans only, blue contours represent an overlaid reference HN correlation.) Proximities seen in (E) are expected on the basis of the monomer structure.77 The peaks labeled in (F) cannot be explained by the monomer fold and hint to major structural rearrangements upon rodlet assembly. Hydrophobin spectra were recorded on deuterated hydrophobin EASΔ15 rodlets, 100% 1H back-exchanged at labile sites in a 1.3 mm rotor at 1 GHz 1H Larmor frequency and 60 kHz MAS. Proton-detected experiments used for obtaining backbone assignments are described in the Supplement.