Structural analysis of alpha-helical proteins from wool using cysteine labelling and mass spectrometry

O’Cualain, Ronan; Sims, Paul F. G.; Carr, Chris

doi:10.1016/j.ijbiomac.2011.05.007

Cited by 5 publications

(8 citation statements)

References 26 publications

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“…It is due to the intramolecular nature of these disulfides that KAPs preferentially swell in the presence of water, as intermolecular bridges will severely limit this process . The observation of multiple KAPs in the second extraction step of our procedure differs from previous results, which found that both reduction and chaotropic disruption were required to solubilize these proteins. The solubilization of KAPs only in the presence of DTT would imply that KAP‐KAP or KAP‐keratin interactions are mainly occurring through the formation of relatively easily accessible disulfide bridges.…”

Section: Discussioncontrasting

confidence: 99%

“…This study led onto the current one which aimed to map the accessibility of cysteine residues within the wool fiber structure, KAPs and keratins were sequentially extracted and labeled, in a procedure similar to that described by O'Cualain et al . The wool was sourced from a Merino ewe from an AgResearch farm and not scoured wool from a commercial supplier.…”

Section: Discussionmentioning

confidence: 99%

“…This was an important starting material as the potential of introducing unwanted modifications of the disulphide bonds due to commercial scouring was avoided. Our approach differed from the one by O'Cualain et al . in two ways: we repeated the exercise five times to examine reproducibility, and we also included an initial step to label the most accessible cysteine residues without prior reduction.…”

Section: Discussionmentioning

confidence: 99%

“…A method described by O'Cualain et al . was modified to sequentially reduce then alkylate five powdered wool samples to determine the accessibility of cysteine residues.…”

Section: Methodsmentioning

confidence: 99%

“…One aim of this investigation was to build on the work described by O'Cualain, et al . to further determine the accessibility of the cysteine residues and their positions in the various regions of the keratins and their reactivity with the matrix proteins.…”

Section: Introductionmentioning

confidence: 99%

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Mapping the accessibility of the disulfide crosslink network in the wool fiber cortex

et al. 2014

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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.

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Section: Discussioncontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…A method described by O'Cualain et al . was modified to sequentially reduce then alkylate five powdered wool samples to determine the accessibility of cysteine residues.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mapping the accessibility of the disulfide crosslink network in the wool fiber cortex

et al. 2014

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A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres

et al. 2021

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Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions 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 detect a greater resolution of statistically nonrandom interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N-or C-terminal domains of these proteins. The most accessible nonrandom cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these interand intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

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Analysis of lecithin treatment effects on the structural transformation of wool fiber using vibrational spectroscopy

Barani

Haji

Maleki

2018

International Journal of Biological Macromolecules

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Structural analysis of alpha-helical proteins from wool using cysteine labelling and mass spectrometry

Cited by 5 publications

References 26 publications

Mapping the accessibility of the disulfide crosslink network in the wool fiber cortex

Mapping the accessibility of the disulfide crosslink network in the wool fiber cortex

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres

Analysis of lecithin treatment effects on the structural transformation of wool fiber using vibrational spectroscopy

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