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.