Mapping the accessibility of the disulfide crosslink network in the wool fiber cortex.
The disulfide bond network within the cortex of mammalian hair has a critical influence on the physical and mechanical characteristics of the fiber. The location, pattern, and accessibility of free and crosslinked cysteines underpin the properties of this network, but have been very difficult to map and understand, because traditional protein extraction techniques require the disruption of these disulfide bonds. Cysteine accessibility in both trichocyte keratins and keratin associated proteins (KAPs) of wool was investigated using staged labeling, where reductants and chaotropic agents were used to expose cysteines in a stepwise fashion according to their accessibility. Cysteines thus exposed were labeled with distinguishable alkylation agents. Proteomic profiling was used to map peptide modifications and thereby explore the role of KAPs in crosslinking keratins. Labeled cysteines from KAPs were detected when wool was extracted with reductant only. Among them were sequences from the end domains of KAPs, indicating that those cysteines were easily accessible in the fiber and could be involved in forming interdisulfide linkages with keratins or with other KAPs. Some of the identified peptides were from the rod domains of Types I and II keratins, with their cysteines positioned on the exposed surface of the α-helix. Peptides were also identified from keratin head and tail domains, demonstrating that they are not buried within the filament structure and, hence, have a possible role in forming disulfide linkages. From this study, a deeper understanding of the accessibility and potential reactivity of cysteine residues in the wool fiber cortex was obtained.
Food & Bio-Based Products, AgResearch, Private Bag 4749, Christchurch, 8140, New Zealand.
Santanu
S
Deb-Choudhury
Jeffrey E
JE
Plowman
Kelsey
K
Rao
Erin
E
Lee
Chikako
C
van Koten
Stefan
S
Clerens
Jolon M
JM
Dyer
Duane P
DP
Harland
D000595:Amino Acid Sequence; D000818:Animals; D003553:Cystine / Q000737:chemistry*; D053564:Keratins, Hair-Specific / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D034561:Sheep, Domestic; D014935:Wool / Q000737:chemistry*
D053564:Keratins, Hair-Specific; D003553:Cystine
keratin associated proteins; mapping; mass spectrometry; trichocyte keratins