INTRODUCTION
Trichocyte keratin intermediate filament proteins (keratins) and the
keratin associated proteins (KAPs) make up the major part of the protein
content of wool fibers 1. The structural domains of
keratins consist of a head (N terminus), central rod and tail
(C-terminus). The central rod region is divided into three highly
conserved α-helical domains, coils 1A, 1B and 2, each with an underlying
structure of heptad repeat sequences separated by short non-helical
linker regions, L1 and L12 2. Coil 2 also has two
additional features: an eleven-residue hendecad repeat region3 at the start of the domain and a ‘stutter’ in which
three of the residues are deleted from the heptad repeat. The heptad
repeats are of the form a-b-c-d-e-f-g in which residues ‘a’ and ‘d’ are
largely apolar. This arrangement results in a hydrophobic stripe running
around the α-helix in a left-handed manner. In the case of the
trichocyte keratins two types exist, the acidic type I and the
neutral-basic type II, their hydrophobic stripes allowing them to
associate to form a coiled-coil heterodimer. This association is also
stabilized by electrostatic interactions between acidic or basic
residues located at positions ‘e’ and ‘g’ in the heptad repeat. In
contrast to the central rod region of the keratins, the head domain is
cysteine rich, has less sequence regularity and, with the exception of a
possible nonapeptide quasi-repeat in type II keratin heads4, no structure has been identified. The tail of both
type I and type II appear to have limited regions that may include some
short α-helix and β-sheet regions, which have the potential to interact
with similar regions in some circumstances 4.
The proteins in the hair fiber are notable for their high cysteine
content, something that distinguishes them from other intermediate
filament forming proteins and also keratins found in epithelial cells5. The trichocyte keratins alone are known to have a
minimum of 20 cysteine residues, most of which are concentrated in the
head and tail domains of the keratins 6. The amount of
cysteine found in the KAPs is even higher, with the so-called high
sulfur proteins (HSPs) having between 20-30 moles% cysteine and the
ultra-high sulfur proteins (UHSPs) having between 30 and 38 moles%
cysteine. It is the interaction between the cysteines in the trichocyte
keratins with those of the KAPs that stabilize the three-dimensional
structures of the keratin intermediate filaments and distinguishes them
from epithelial keratins 2. These interactions are
thought to be the main driver of fiber functionality and important in
reduction-then-oxidation processes such as stretch set in the
manufacture of wool garments 7. Removal of cysteine
from wool fibers through the conversion of cysteine to cysteic acid, by
for example, oxidative bleaching with hydrogen peroxide to whiten wool,
is known to impair process like stretch set considerably7.
Keratin head domains are thought to be important for intermolecular
interactions with KAPs, largely assumed to be disulfide bonding. Recent
co-immunoprecipitation studies have demonstrated an interaction between
the head domain of human K86 with KAP2.1 8, while
Western blot studies have demonstrated an interaction between the head
domain of K85 and KAP8.1 8. However, studies have
indicated that approximately 97.5% of cysteines in the KAPs are
involved in intramolecular bonding 9. Early proteomic
studies on wool using two-dimensional electrophoresis suggested that
only a few discrete cysteine residues in the KAPs were affected by
treatment with hydrogen peroxide 10. Differential
accessibility of the cysteines in single samples of wool has also been
investigated by mass spectrometry protocols involving reduction followed
by alkylation and then extraction 11. To further
investigate non-random cysteine-accessibility we undertook a study, on
which the current study builds, in which wool was subjected to a process
of incremental labelling whereby reductants and chaotropes were used in
stages to reductively expose and label cysteines in the fiber in a
stepwise fashion according to their accessibility, after which the
labelled peptides were extracted and identified by proteomics, a process
repeated five times at each stage 12. In this study
when we refer to residues, we refer exclusively to these with a
non-random accessibility. From this it became apparent that the most
readily accessible cysteines were within the head or tail domains of the
keratins. Given that at least 50% of the head and tail domains of the
trichocyte keratins lie along the inner core of the strongly apolar
intermediate filament 13, where reducing agents would
be less effective, this would suggest that these accessible cysteines
are most likely on the outer surface of the filament. In contrast, no
conclusion could be drawn on the cysteine accessibility of the KAPs
because, under the conditions investigated, the KAPs did not display the
sequential accessibility of the keratins, all repeatably non-random
cysteine labelling occurring in one stage/step. One limitation of our
earlier study on the effect of reductant on cysteine accessibility was
that the lowest concentration of DTT examined was 20 mM12. Another study, however, has shown that the various
components of the KAPs exhibit a differential extractability when
sequentially exposed to concentrations of DTT between 5 and 20 mM14. It was for
this reason that we extended this approach to study the effect of these
lower concentrations of reductant not only to understand the effect on
the accessibility of the cysteines in the KAPs in the mature
wool fiber but also to examine the accessibility of the cysteines in
keratins at these concentrations with the view to gaining an insight as
to how the disulfide bridges may form between keratins and KAPs.