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.