Fungal Fibrils: Application of the Amyloid Polymer Structure by Fungi - The Functional Fold: Amyloid Structures in Nature - page 42

straight, and unbranched in the electron microscope. The rodlets
are 50-120 Å wide and appear to be composed of protofilaments
that are approximately 2.5 Å wide. The rodlets form monolayers that
are approximately 70-100 Å thick.

10,33,34

Given the sequence and
physicochemical similarities of the Class I hydrophobins, it seems
likely that these amyloid-like features will be common to all rodlets
formed by this group of hydrophobins.
Preparations of unaligned EAS rodlets (produced by vortexing
and then harvested by centrifugation) display a cross-

β

X-ray fibre

diffraction pattern (Fig. 3.4b). The dominant reflections in the

pattern are at 4.8 and 10-12 Å, consistent with the inter-strand
and inter-sheet spacings present in the

35

β

-sheet structure. The
X-ray diffraction pattern obtained from a monolayer rodlet film
formed by drying EAS rodlets in a magnetic field displays some
preferential orientation of the inter-strand spacing and an additional
perpendicular reflection at approximately 27 Å, which matches the
diameter of the monomer. These reflections are consistent with
the hydrogen bonding between strands lying in the direction of the
rodlet long axis. There is no prominent inter-sheet spacing in this
pattern, probably because this is a monolayer sample and so the
inter-sheet spacing is not repeated in this direction.
Similar to amyloid fibrils, Class I hydrophobin rodlets display
yellow-green birefringence when stained with the dye Congo red
and viewed between cross-polarizers; they also bind to Thioflavin-T,
and the dye exhibits enhanced fluorescence when bound to rodlets
(Fig. 3.4c,d).

31-33,36

The monomeric, unassembled forms of the
hydrophobins do not bind to these dyes, indicating that a structural
change must occur upon rodlet formation to generate the extended,
stacked

-sheet structure. Taken together, the nature of the X-ray
fibre diffraction pattern and the specific interactions with Congo red
and Thioflavin-T suggest that Class I hydrophobin rodlets have an
underlying ordered

β

β

-sheet core.
Amyloid fibrils are composed of different numbers of sub-
filaments. The number and arrangement of the sub-filaments
appears to differ depending on the nature of the polypeptide forming
the fibril and also the conditions under which fibrillogenesis occurs.
Twisting of fibrils is also often observed. However, amyloid fibrils
do not generally associate laterally in a specific or organized fashion
over the length of the fibril. Rodlets, by contrast, show specific
and directed lateral assembly and generate monolayers that are
amphipathic. This implies that, in addition to the forces that drive