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Mol Cell Proteomics DOI:10.1074/mcp.RA119.001325

Chasing Tails: Cathepsin-L Improves Structural Analysis of Histones by HX-MS.

Publication TypeJournal Article
Year of Publication2019
AuthorsPapanastasiou, M, Mullahoo, J, DeRuff, KC, Bajrami, B, Karageorgos, I, Johnston, SE, Peckner, R, Myers, SA, Carr, SA, Jaffe, JD
JournalMol Cell Proteomics
Volume18
Issue10
Pages2089-2098
Date Published2019 10
ISSN1535-9484
Abstract

The N-terminal regions (tails) of histone proteins are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications present on these regions contribute to transcriptional regulation. Considering their biological significance, relatively few structural details have been established for histone tails, mainly because of their inherently disordered nature. Although hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed in this context, presumably because of the poor N-terminal coverage provided by pepsin. Inspired from histone-clipping events, we profiled the activity of cathepsin-L under HX-MS quench conditions and characterized its specificity employing the four core histones (H2A, H2B, H3 and H4). Cathepsin-L demonstrated cleavage patterns that were substrate- and pH-dependent. Cathepsin-L generated overlapping N-terminal peptides about 20 amino acids long for H2A, H3, and H4 proving its suitability for the analysis of histone tails dynamics. We developed a comprehensive HX-MS method in combination with pepsin and obtained full sequence coverage for all histones. We employed our method to analyze histones H3 and H4. We observe rapid deuterium exchange of the N-terminal tails and cooperative unfolding (EX1 kinetics) in the histone-fold domains of histone monomers in-solution. Overall, this novel strategy opens new avenues for investigating the dynamic properties of histones that are not apparent from the crystal structures, providing insights into the structural basis of the histone code.

DOI10.1074/mcp.RA119.001325
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/31409669?dopt=Abstract

Alternate JournalMol. Cell Proteomics
PubMed ID31409669
PubMed Central IDPMC6773551
Grant ListU54 HG008097 / HG / NHGRI NIH HHS / United States