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are often those that display enhanced conformational dynamics
and which populate partially structured intermediates.
Like
many amyloid-forming proteins, the Class I hydrophobins appear
to undergo some level of conformational change when rodlets form,
whereas the Class II hydrophobins do not.
44
45
Techniques such as circular dichroism spectropolarimetry (CD)
and Fourier transform infrared spectroscopy (FTIR) have been used
to follow the structural changes which occur during rodlet formation
by Class I hydrophobins. The CD spectrum of soluble SC3 indicates
that the protein has elements of
β
-sheet structure, together with
some random coil. When colloidal Teflon
TM
is added to a solution
of SC3, spectral features indicative of
α
-helical structure appear.
These
-helical elements may be relevant to the polymerization
process in SC3 since an on-pathway intermediate has been proposed
to be important in rodlet formation.
α
46-48
The CD spectra from the
soluble forms of other Class I hydrophobins including EAS, SC4,
and ABH3 indicate that they all contain varying amounts of
β
-sheet
30,33,49
and random coil structure.
However, in all cases, a significant
increase in
-sheet structure is detected when rodlets form, which is
also commonly observed upon amyloid formation (Fig. 3.7).
The FTIR spectrum of soluble SC3, dried onto a germanium plate,
is consistent with the presence of a mixture of secondary structure
β
.
Deconvolution and curve fitting analysis of the single major amide I
band at 1636 cm
46
−
1
suggests that soluble SC3 is composed of 23%
α
β
β
-turn, with the remaining 20% being
random coil. Drying down of SC3 onto a silanized germanium plate,
which increases the surface hydrophobicity, changed the profile
to one with mainly
-helix, 41%
-sheet, and 16%
-sheet contributions, suggesting
that the assembling SC3 intermediates might contain helical
structure. The final assembled rodlet form of SC3 is
α
-helix and
β
β
46,47
-sheet rich,
33
as is the rodlet form of EAS.
Polarization-modulated infrared
reflectance adsorption spectroscopy (PM-IRRAS) indicates that SC3
localizes to the surface of the solution rapidly, where it undergoes
a conformational change to a mainly
47
This form of
spectroscopy is also able to report on the orientation of secondary
structure elements relative to the incident surface. In the case of SC3
rodlets, the nature of the amide signal indicates that hydrogen bonds
are oriented preferentially parallel to the air-water interface. This
shows that the plane of the
β
-sheet form.
β
-sheet structure, in which the hydrogen
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