DISCUSSION
New highly accessible disulfides were
identified
In combination with our earlier step-based approach to cysteine
accessibility, the findings here help give a more complete picture of
disulfide accessibility in the wool fiber because between the two
studies we have seven levels of reducing and/or chaotropic intensity
(Figure 5).
Keratin associated
proteins
In our earlier study of cysteine accessibility the cysteines in the KAPs
most affected tended to be found in the central region of the proteins
and usually close to a proline residue 12. The HSPs
and UHSPs in particular are notable for having a pentapeptide repeat of
the structure of the following forms: A1, C-C-Q-P-X or A2, C-C-R-P_X
and B, C-C-X-S/T-S/T, in which the presence of a cysteine close to a
proline residue was thought to confer a conformation in the protein that
may have made these cysteines more accessible 312. In contrast, in this study at 5 mM DTT the only
consistently modified cysteines were located at either the N- or
C-termini of the three HSPs. Raising the DTT concentration to 10 mM DTT
not only exposed more N- and C-terminal cysteines in the KAPs but also
some of the centrally located cysteines in these proteins.
Keratins
This study has provided evidence that the majority of the accessible
cysteines were found in the head and tail domains of the keratins at
concentrations of DTT lower than the concentration used previously12. This is particularly true for the major type II
keratins, K81, K83, K85 and K86, where consistently modified cysteines
were only found in the head and tail domains (Figure 4, Table 2). In the
case of the type I keratins consistently modified cysteines were found
in both the head and tail domains in K34 and K35 but only in the tail
domain of K31 and K33a, (Figure 4, Table 2). In addition, some cysteines
in coil 1B were also accessible in K31 and in the L1 linker of K34 at 10
mM DTT, as well as cysteines close to the tail domain in coil 2 in K31,
K33a, K33b, K34 and K35.
Studies of trichocyte keratin structure have indicated that during the
process of assembly of keratins into coiled-coil dimers, tetramers,
unit-length filaments and intermediate filaments, half of the head and
tail domains are thought to protrude from the surface of this filament
where they are free to interact with the KAPs 1315. Thus, the labelled cysteines in the head domains
of K34, K35, K81, K83 and K85 are possible sites for keratin-KAP
disulfide crosslinks. Likewise, the cysteines in the C-terminus of K31,
K33a, K34, K81, K83, K85 and K86 are also possible sites for keratin-KAP
disulfide crosslinks, although repeatably accessible cysteines close to
the tail domain coil 2B (noted above) are also potential partners.
Keratin rod domain and tetramer
linkages
Studies of disulfide crosslinking in human and mouse epithelial keratins
have shown the locations of a number of intermolecular crosslinks in the
coil 2 region between pairs of heterodimers 16. For
instance in the reduced form of the mouse and human epithelial type I
keratin K10, a cysteine in coil 2B at position 2B-62 (position 2-89 in
the hendecad-heptad motif of coil 2) has previously been identified as
the site of an intermolecular heterodimer disulfide crosslink with
another 2B-62 (2-89) cysteine residue of K10 16. In
this study, an accessible cysteine was found near the stutter in
position 2-90 in coil 2 of K31 (Figure 3, Table 2), which would mean
that this cysteine is possibly in a good orientation to crosslink with
cysteine in the same position in another type I keratin because the
A22 axial alignment of trichocyte keratins is unchanged
between the reduced and oxidized states and also identical to the
reduced form of epithelial keratins (Figure 6). However, such an
interaction would only be possible if the axial separation of the two
cysteines is appropriate and if the stereochemistry is correct. One
consequence of this is that it may involve some local unwinding or
distortion to allow the energetically favorable disulfide bonds to form17. A further readily reducible cysteine was detected
at position 2-132 in coil 2 of K31 and K33a (position 2B-105 in the
2A-L2-2B coil system) (Figure 3, Table 3) and this has also been
identified as the site of a disulfide bond between 2-132 in K31 and 2-49
in K83 18. One cysteine at position T-5 in the tail
domain of K83 and K86 is also in a similar position to a cysteine at
position T-4 human and mouse epithelial K1 that is involved in
keratin-keratin crosslinking with a cysteine close to the C-terminus of
the head domain in type I keratins 16, meaning that
this could be another site of keratin-keratin crosslinking in trichocyte
keratins. Finally, in coil 1 there are two readily reducible cysteines,
L1-9 in K34 and 1B-87 in K31. In mouse trichocyte keratins these
residues have been identified as possibly being involved in another
disulfide crosslinking site between heterodimers in the tetramer18 19. Thus, the ready accessibility
of these cysteines in the keratins suggests that these are potential
sites of disulfide breakage as the fibers age and are subject to
oxidative damage from such factors as sunlight, heat or chemical
treatments.