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\begin{document}

\title{A detailed mapping of the readily accessible disulfide bonds in the
cortex of wool fibers}



\author[1]{Jeffrey Plowman}%
\author[2]{Rachel Miller}%
\author[2]{Ancy Thomas}%
\author[2]{Anita Grosvenor}%
\author[1]{Duane Harland}%
\author[3]{Santanu Deb-Choudhury}%
\affil[1]{AgResearch Ltd.,}%
\affil[2]{AgResearch Ltd}%
\affil[3]{AgResearch Ltd.}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
One way in which trichocyte keratin intermediate filament proteins
(keratins) and keratin associated proteins (KAPs) differ from their
epithelial equivalents is in their higher levels of cysteine residues.
Interactions between these cysteine residues within a mammalian fiber,
and the putative regular organization of interactions (i.e., types of
disulfide bond) are likely important for defining fiber mechanical
properties, and thus biological functionality of hairs. Here we extend a
previous study of cysteine accessibility under different levels of
exposure to reducing compounds to explore a finer set of levels
associated with interactions between keratins and KAPs. We found that
most of the cysteines in the KAPs were close to either the N- or C-
terminal domains of these proteins. The most accessible cysteines in
keratins were present in the head or tail domains indicating their
function in readily forming intermolecular bonds with KAPs. Some of the
more buried cysteines in keratins were discovered either close to or
within the rod region in positions previously identified in human
epithelial keratins as being involved in crosslinking between the
heterodimers of the tetramer. Our present study therefore provides a
deeper understanding of the accessibility of disulfides especially in
keratins and thus proves that there is some specificity to the disulfide
bond interactions leading to these intermolecular bonds stabilizing the
fiber structure.%
\end{abstract}%



\sloppy


\section*{INTRODUCTION}

{\label{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 \textsuperscript{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 \selectlanguage{greek}α-\selectlanguage{english}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 \textsuperscript{2}. Coil 2 also has two
additional features: an eleven-residue hendecad repeat
region\textsuperscript{3} 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 \selectlanguage{greek}α-\selectlanguage{english}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
heads\textsuperscript{4}, no structure has been identified. The tail of
both type I and type II appear to have limited regions that may include
some short \selectlanguage{greek}α-\selectlanguage{english}helix and \selectlanguage{greek}β-\selectlanguage{english}sheet regions, which have the potential to
interact with similar regions in some circumstances \textsuperscript{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
cells\textsuperscript{5}. 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 \textsuperscript{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 \textsuperscript{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 \textsuperscript{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
considerably\textsuperscript{7}.

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 \textsuperscript{8}, while
Western blot studies have demonstrated an interaction between the head
domain of K85 and KAP8.1 \textsuperscript{8}. However, studies have
indicated that approximately 97.5\% of cysteines in the KAPs are
involved in intramolecular bonding \textsuperscript{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 \textsuperscript{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 \textsuperscript{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 \textsuperscript{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 \textsuperscript{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
mM\textsuperscript{12}. 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
mM\textsuperscript{14}. 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.

\section*{METHODS AND MATERIALS}

{\label{methods-and-materials}}

\subsection*{Materials}

{\label{materials}}

Ammonium bicarbonate, ethylenediaminetetraacetic acid (EDTA), formic
acid, methanol, sodium phosphate and urea were obtained from Merck
(Darmstadt, Germany). Dithiothreitol (DTT), iodoacetic acid (IAA), and
iodoacetamide (IAM) were obtained from Sigma (St Louis, MO, USA). Bovine
serum albumin (BSA) was obtained from ICPBio, Auckland, NZ). Acrylamide
was obtained from Bio-Rad (Hercules, CA, USA). LC/MS-grade acetonitrile
was obtained from Fisher Scientific (Pittsburg, USA). TPCK-trypsin was
obtained from Promega (Fitchburg, MI, USA). LC/MS-grade water was
obtained from Fluka. Empore disks were obtained from Supelco
(Bellefonte, PA, USA).

\subsection*{Preparation of merino
wool}

{\label{preparation-of-merino-wool}}

Merino wool with fibers of \textasciitilde{}18 \selectlanguage{greek}μ\selectlanguage{english}m diameter were sourced
from a ewe from a farm in mid-Canterbury, New Zealand. The wool was
detipped by removing the top third of the fiber and the extraneous dirt
and sebum removed by subjection to agitation in an ultrasonic bath in
ultrapure water for 15 mins three times. After this the wool fibers were
washed sequentially twice in dichloromethane, twice in ethanol, then
twice in water before air drying overnight. After this the wool was cut
into snippets of less than 1 mm length with scissors.

\subsection*{Sequential alkylation with IAM, acrylamide and
IAA}

{\label{sequential-alkylation-with-iam-acrylamide-and-iaa}}

The method of stepwise reduction and labelling of cysteines is
summarized in Figure 1, where cysteines in the wool fiber are
progressively reduced by increasing the concentration of DTT. This
experiment was split into three steps involving the reduction of five
independent samples with 5 mM, 10 mM and 15 mM of DTT, followed by
alkylation with one of three different reagents: iodoacetamide (IAM),
acrylamide and iodoacetic acid (IAA) to determine at which step the
cysteine group became accessible to the labelling reagent.

Prior to commencing the experiment, a control run was made with a sample
of the snipped wool in the absence of DTT. This was incubated in the
base buffer solution only (100 mM sodium phosphate buffer (pH 7.4), 1 mM
EDTA) for 24 h followed by labelling with 2 mM IAM in base buffer for 30
min in the dark. The supernatant was collected after the extract was
centrifuged at 44,000 g for 15 min. This was Step 0 (Figure 1).

In the first step, 20 mg of cleaned wool was incubated in 1.5 mL of the
base buffer containing 5 mM DTT for 24 h followed by labelling with 2 mM
IAM in the base buffer for 30 min in the dark. The supernatant was
collected after the extract was centrifuged at 44,000 g for 15 min.
Residual extraction buffer was removed from the pellet by vortexing for
1 min in wash buffer (10 mM sodium phosphate buffer (pH 7.4), 1 mM
EDTA), followed by centrifugation at 44,000 g for 15 min. This was
repeated three times and these washes discarded. After this the pellet
was suspended in wash buffer and centrifuged. Mass spectrometry was used
to detect the presence of residual IAM. This was performed by taking 60
\selectlanguage{greek}µ\selectlanguage{english}L of the supernatant from the washed wool pellet and added to 10 \selectlanguage{greek}µ\selectlanguage{english}L of
0.2 mg/mL of reduced BSA and vortexed in the dark for 30 min at room
temperature. This BSA was digested with trypsin and the resulting digest
analyzed for alkylation of the BSA peptides by mass spectrometry. If no
alkylation was detected in the BSA peptides, indicating the absence of
residual IAM, the washed pellet was then used in the next step for
further analysis.

In the second step the washed wool pellet was incubated in the base
buffer solution containing 10 mM DTT for 24 h. This was followed by
labelling with 350 mM acrylamide in the base buffer for 24 h in the
dark. The supernatant was then collected after centrifugation and the
pellet washed three times with wash buffer and checked for residual
alkylating reagent, as described above, before proceeding to the next
step.

In the third step the washed wool pellet was incubated in the base
buffer containing 15 mM DTT for 24 h. This was followed by labelling
with 2 mM iodoacetic acid in base buffer for 30 min in the dark, after
which the extract was centrifuged and the supernatant collected, while
the pellet was discarded.

\subsection*{Preparation for Mass
Spectrometry}

{\label{preparation-for-mass-spectrometry}}

Each extract was digested with TPCK-trypsin at an enzyme-substrate ratio
of 1:50 in 50 mM ammonium bicarbonate:AcN (9:1) at 37 \selectlanguage{ngerman}°C for 18 h. The
peptide digest was vortexed for 3 h in the presence of Empore\selectlanguage{english} disks
that had been prewetted with AcN and methanol. Following this the
Empore\selectlanguage{english} disks were washed with 0.1\% TFA and extracted with 75\% AcN in
0.1\% trifluoroacetic acid. After drying the extracts were resuspended
in 50 \selectlanguage{greek}μ\selectlanguage{english}L of 0.1\% trifluoroacetic acid for further analysis.

\subsection*{Mass spectrometry
analysis}

{\label{mass-spectrometry-analysis}}

LC-MS/MS was performed on a nanoflow UPLC system (Dionex Ultimate 3000)
coupled to an Impact II mass spectrometer equipped with a CaptiveSpray
source (Bruker Daltonik, Bremen, Germany). For each sample, 1 \selectlanguage{greek}μ\selectlanguage{english}L of the
sample was loaded on a C18 PepMap100 nano-Trap column (200 \selectlanguage{greek}μ\selectlanguage{english}m x 2 cm) at
a flow rate of 5 \selectlanguage{greek}μ\selectlanguage{english}L/min. The trap column was then switched in line with
the analytical column ProntoSIL C18AQ (100 \selectlanguage{greek}μ\selectlanguage{english}m ID x 150 mm 3 micron 200
\selectlanguage{ngerman}Å). The reverse phase elution gradient was from 2\% to 20\% to 45\% B
over 60 min, total 84 min at a flow rate of 1000 nL/min. Solvent A was
LCMS-grade water with 0.1\% formic acid; solvent B was LC-MS-grade AcN
with 0.1\% formic acid.

Samples were measured in automatic MS/MS mode, with a mass range of m/z
150--2200. Acquisition speed was 5 Hz in MS and 1-50 Hz in MS/MS mode
depending on precursor intensity. Ten precursors were selected in the
m/z 300-1250 range, with a preference for doubly or triply charged
peptides. The analysis was performed in positive ionization mode with a
dynamic exclusion of 60 sec.

\subsection*{Data Analysis}

{\label{data-analysis}}

Following the LC-MS run, the QTOF data were searched using Peaks Studio
8.5 (Bioinformatics Solutions Inc). The raw data were refined by a
built-in algorithm and protein/peptides were identified using the
following parameters: a precursor mass tolerance of 10 ppm; fragment
mass tolerance of 0.05 Da, a taxonomy of \emph{Ovis aries} with an in
house Sheep\_uniprot\_ovine\_2016\_10\_plus\_keratins database. This
database was derived from the Uniprot 2016\_10 database, augmented by 54
AgResearch sequences from EST contigs, annotated by BLAST searching
against the NCBI database and keratin sequences from the NCBInr
non-redundant database. Trypsin was chosen as the proteolytic enzyme and
up to 2 mis-cleavages were allowed. Carbamidomethyl (C), propionamide
(C) and carboxymethylation (C) were chosen as variable modifications. A
false discovery rate (FDR) of 0.5\% was used for peptide identification
in Peaks. In addition, the Peaks post-translational modification (PTM)
A-score was set to 30, de novo only ALC \textgreater{} 85\% and only
proteins with a minimum of 2 unique peptides identification were
included. As the keratin-family of proteins is known to have highly
similarly in protein sequences, the quality of identification of all
peptides was checked manually.

In the final analysis a peptide sequence containing the same labelled
cysteine was only considered to be significant if it was found in four
of the five repeats. This was because in our previous study it was
established that only peptide sequences with the same labelled cysteine
observed in at least four out of five repeats had a significance level
of \emph{P \textless{}0.05} \textsuperscript{12}. That is, any peptide
with less than four observations was considered to have non-repeatable,
and therefore random, accessibility.

\section*{RESULTS}

{\label{results}}

\subsection*{Sequential labelling of
cysteines}

{\label{sequential-labelling-of-cysteines}}

In order to determine the relative accessibility of cysteines in wool
the keratins and KAPs were subjected to a process of sequential
alkylation as the concentration of DTT was raised in stepwise fashion
from 0 to 15 mM.

A total of 38 peptides were detected and identified in the mass spectrum
of protein extracts in the control run, involving wool that had not been
treated with a reductant. Of these, two were from the epithelial keratin
K10 and the rest were from either actin or serum albumin. A number of
cysteines in the peptides were labelled with IAM but only in those from
serum albumin.

In contrast cysteine labelling of keratins and KAPs was only detected
when the wool was incubated with DTT. Among the labelled cysteines were
a number identified in our earlier study \textsuperscript{12},
specifically: in the tail domain of K31, T-42 and T-45; in coil 1B of
K31 and K33b, 1B-87; the head domain of K35, H-36; H-24 and H25 in K81
(H-29 and H30 in K83); and H61 and T-24 in K85.

In the first step, when a DTT concentration of 5 mM was used to reduce
the proteins, eight peptides from four keratins and three KAPs
containing IAM-labelled cysteines were identified in at least four out
of the five biological repeats (Figures 2-4, Table 1). Interestingly,
all the keratin peptides identified were labelled on either the head or
tail domain. In the case of the type I keratins one peptide from the
tail region of K31 was identified on which four cysteine residues were
labelled. Peptides from three Type II keratins were also found to have
IAM-labelled cysteines. For K83 this involved two peptides from the head
domain of which three cysteines were found to be labelled. In the case
of K85 a peptide containing one cysteine from its tail domain was
labelled, while a peptide containing one cysteine from the tail domain
of K86 was also labelled. IAM-labelled peptides were identified from
three HSPs, specifically cysteines close to the N-terminus of KAP2.3 and
the cysteines close to the C-terminus of KAP7.1, KAP11.1 and KAP13.1
(Figure 2, Table 1).

In the second step, proteins were unraveled further by reduction with 10
mM DTT and then labelled with acrylamide (Figures 2-4, Table 2). This
resulted in the identification of a total of 62 cysteine-labelled
peptides, of which 20 were from keratins and 42 from KAPs. Of the KAPs,
65\% of were classed as HSPs, 16\% UHSPs and the rest high
glycine-tyrosine proteins (HGTPs). In the case of the type II keratins
all the newly characterized peptides in this step (and hence cysteines)
were either located in the head or tail domains (Figure 4). In contrast,
in the type I keratins one acrylamide-labelled cysteine was identified
in a peptide from coil 1B and another from coil 2, while an
acrylamide-labelled cysteine was identified in a peptide from the L1
linker in K34 (Figure 3). No modified cysteines were found in the
hendecad regions \textsuperscript{3}. As found
previously\textsuperscript{12} around 90\% of the labelled cysteines in
the HSPs and UHSPs are found close to a proline residue in the sequence
and over half within 20 residues of the N- and C-terminus of the
proteins. Specifically these involved cysteines at the N-terminus of
KAP3.3, KAP3.4 and KAP7.1, and cysteines close to the C-terminus of
KAP31, KAP3.4, KAP4.2, KAP4.5, KAP9.2, KAP9.8, KAP10.11, KAP10.12,
KAP12.2, KAP19.5 and KAP26.1 (Figure 2, Table 2). At the same time a
number of cysteines were detected from the more central regions of some
of the KAPs, among them KAP2.3, KAP3.4, KAP4.2, KAP6.1, KAP6.4, KAP10.4,
KAP11.1,KAP13.1, KAP16.1-like, KAP19.3, KAP24.1 and KAP26.1.

In the third step very few IAA-labelled peptides were identified in the
final alkylation step. Of the two keratin peptides identified the one
from K31 was labelled in the coil 2 domain while that from K35 was
labelled in the head domain (Figure 3, Table 3). Two KAPs were
identified in this stage, one being KAP 6.1, an HGTP, and the second
being from the HSP KAP13.1-like (Figure 2, Table 3).

\subsection*{Validation of the labelling
experiments}

{\label{validation-of-the-labelling-experiments}}

To determine if the large increase in peptides identified in step 2 and
the subsequent drop in peptide numbers in step 3 was due to the longer
labelling time used with acrylamide the entire process was repeated
using IAM in all steps. However, a similar pattern was observed when
comparing the proportion of peptides identified between each of the
steps. This led to the conclusion that the alkylation reagent used was
not influencing the efficiency of the labelling and thus peptide
identification.

\section*{DISCUSSION}

{\label{discussion}}

\subsection*{New highly accessible disulfides were
identified}

{\label{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).

\subsection*{Keratin associated
proteins}

{\label{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 \textsuperscript{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 \textsuperscript{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.

\subsection*{Keratins}

{\label{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
previously\textsuperscript{12}. 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 \textsuperscript{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.

\subsection*{Keratin rod domain and tetramer
linkages}

{\label{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 \textsuperscript{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 \textsuperscript{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
A\textsubscript{22} 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
form\textsuperscript{17}. 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 \textsuperscript{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 \textsuperscript{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
tetramer\textsuperscript{18} \textsuperscript{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.

\section*{CONCLUSIONS}

{\label{conclusions}}

This study of the cysteine accessibility in the keratins and KAPs of the
wool cortex has revealed the following:

\begin{itemize}
\tightlist
\item
  Repeatably accessible (non-random) cysteines found in the head and
  tail domains in the type I and II keratins are likely to be on the
  surface of the intermediate filament and, hence, available for
  interactions with KAPs.
\item
  Two accessible cysteines were found in coil 2 in the type I keratin
  K31 which are potentially involved in crosslinks with other keratins
  in the filament. One cysteine in position 2-90 in a type I keratin is
  possibly in a good orientation to be involved in a crosslink with
  another cysteine also at position 2-90 in a second type I keratin,
  while a second cysteine at position 2-132 in a type I keratin has also
  been identified as the site of another potential keratin-keratin
  crosslink.
\item
  One accessible cysteine at position T-5 in the tail domain of K83 and
  K86 is in a position where it could be involved in crosslinking with a
  cysteine close to the C-terminus of the head domain of type I
  keratins.
\item
  Cysteines at positions L1-9 of K34 and 1B-87 of K31 are most probably
  linked via a disulfide interaction.
\item
  The ready accessibility of the cysteines involved in disulfide
  interactions between keratins in the intermediate filament point to
  how oxidative stress on the fiber brought on by environmental factors
  has the potential to affect the integrity of the filament itself.
\item
  The bulk of the most readily accessible cysteines in the KAPs were
  found close to either the N- or C-terminal domains in these proteins.
\end{itemize}

\section*{Acknowledgements}

{\label{acknowledgements}}

The authors are grateful to Erin Lee for preparing the samples for mass
spectrometry, Evelyne Maes for helpful comments during the preparation
of the manuscript and Professor David Parry of Massey University for
helpful discussions. The work was funded by the Wool Research
Organisation of New Zealand (WRONZ) and the Ministry of Business
Innovation and Employment through the New Materials from New Zealand
Wool partnership program via Wool Industry Research Limited.

\textbf{Figures}

\textbf{Figure 1.} Schematic summary of the sample preparation
methodology steps.

\textbf{Figure 2:} KAP sequences containing accessible cysteines
identified in stage 1 (Bold), Step 2 (Bold underlined) and Step 3 (Bold
Italicized).

\textbf{Figure 3.} Keratin type I sequences containing accessible
cysteines identified in Step 1 (Bold), Step 2 (Bold underlined) and Step
3 (Bold Italicized), labelled cysteines are in red.

\textbf{Figure 4.} Keratin type II sequences containing accessible
cysteines identified in Step 1 (Bold), Step 2 (Bold underlined) and Step
3 (Bold Italicized), labelled cysteines are in red.

\textbf{Figure 5.} A summary of the accessibility of cysteines in type I
and II keratins. The two chains (I and II) are indicated by different
shades of the same color. K31 and K85 are examples that demonstrate a
general trend.

\textbf{Figure 6.} A schematic representation of the disulfide
crosslinks between a pair of antiparallel molecules in the oxidized
tetramer. The disulfide crosslinks are between cysteines at positions
L1-9 and 1B-87 in type I keratins in the A\textsubscript{11} alignment
and two cysteines at position 2-90 in type I keratins in the
A\textsubscript{22} alignment. Two other cysteines (2-132 and T-5)
thought to be involved in crosslinking are also marked on the diagram.
The two chains (I and II) are indicated by different shades of the same
color.

\section*{References}

{\label{references}}

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