Stepwise Characterization of the Thermodynamics of Trichocyte Intermediate Filament Protein Supramolecular Assembly
Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.Journal of Molecular Biology (Impact Factor: 4.33). 03/2011; 408(5):832-8. DOI: 10.1016/j.jmb.2011.03.034
Trichocyte intermediate filament protein (IFP) is a heterodimeric complex that plays a pivotal role in the hair shaft for its mechanical strength, hair shape, and so on. Trichocyte IFP consists of acidic-type IFP and basic-type IFP, and the well-studied supramolecular assembly process of the complex occurs via the following steps: dimer formation, tetramer formation, formation of the lateral 32mer, and the elongation of the 32mer. Among these interactions, only the dimer formation, owing to coiled-coil interaction, has been described in detail; the nature of other interactions remains unspecified. For each assembly step, we report interaction isotherms obtained by means of isothermal titration calorimetry at various urea and NaCl concentrations. Decreasing the urea concentration generally promotes protein refolding, and we therefore expected to observe endothermic interactions owing to the refolding process. However, exothermic interactions were observed at 4 and 2 M urea, along with various characteristic endothermic interactions at the other urea concentrations as well as NaCl titration. The thermal responses described herein enabled us to analyze the protein supramolecular assembly process in a stepwise manner.
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ABSTRACT: Since their first finding in wool 50 years ago, keratin-associated proteins (KAPs), which are classified into 3 groups; high sulfur (HS) KAPs, ultra high sulfur (UHS) KAPs, and high glycine-tyrosine (HGT) KAPs, have been the target of curiosity for scientists due to their characteristic amino acid sequences. While HS and UHS KAPs are known to function in disulfide bond crosslinking, the function of HGT KAPs remains unknown. To clarify the function as well as the binding partners of HGT KAPs, we prepared KAP8.1 and other KAP family proteins, the trichocyte intermediate filament proteins (IFP) K85 and K35, the head domain of K85, and the C subdomain of desmoplakin C-terminus (DPCT-C) and investigated the interactions between them in vitro. Western blot analysis and isothermal titration calorimetry (ITC) indicate that KAP8.1 binds to the head domain of K85, which is helically aligned around the axis of the intermediate filament (IF). From these results and transmission electron microscopy (TEM) observations of bundled filament complex in vitro, we propose that the helical arrangement of IFs found in the orthocortex, which is uniquely distributed on the convex fiber side of the hair, is regulated by KAP8.1. Structure-dependent binding of DPCT-C to trichocyte IFP was confirmed by Western blotting, ITC, and circular dichroism. Moreover, DPCT-C also binds to some HGT KAPs. It is probable that such bidirectional binding property of HGT KAPs contribute to the mechanical robustness of hair.
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ABSTRACT: The disulfide bond network within the cortex of mammalian hair has a critical influence on the physical and mechanical characteristics of the fibre. 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 labelling, where reductants and chaotropic agents were used to expose cysteines in a stepwise fashion according to their accessibility. Cysteines thus exposed were labelled with distinguishable alkylation agents. Proteomic profiling was used to map peptide modifications and thereby explore the role of KAPs in crosslinking keratins. Labelled 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 fibre and could be involved in forming inter-disulfide linkages with keratins or with other KAPs. Some of the identified peptides were from the rod domains of Type I and Type 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 fibre cortex was obtained. This article is protected by copyright. All rights reserved.
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