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

Area of Science:

• Biochemistry
• Structural Biology
• Epigenetics

Background:

• Histone N-terminal tails are crucial for chromatin organization and epigenetic regulation.
• Their dynamic and disordered nature presents challenges for structural analysis.
• Existing methods like hydrogen/deuterium exchange mass spectrometry (HX-MS) have limitations in histone N-terminal coverage.

Purpose of the Study:

• To develop a novel HX-MS strategy for detailed analysis of histone tail dynamics.
• To characterize the specificity and utility of cathepsin-L for histone N-terminal peptide generation.
• To investigate the solution dynamics of histone monomers, specifically H3 and H4.

Main Methods:

• Profiling cathepsin-L activity under HX-MS conditions with core histones (H2A, H2B, H3, H4).
• Characterizing cathepsin-L specificity and cleavage patterns.
• Developing a comprehensive HX-MS method combining pepsin and cathepsin-L for full histone sequence coverage.
• Analyzing deuterium exchange kinetics and unfolding of histone H3 and H4 monomers in solution.

Main Results:

• Cathepsin-L demonstrated substrate- and pH-dependent cleavage, generating suitable N-terminal peptides for H2A, H3, and H4.
• The combined HX-MS method achieved full sequence coverage for all core histones.
• Analysis of histones H3 and H4 revealed rapid deuterium exchange in N-terminal tails.
• Cooperative unfolding (EX1 kinetics) was observed in the histone-fold domains of histone monomers in solution.

Conclusions:

• The novel HX-MS strategy using cathepsin-L and pepsin enables detailed investigation of histone tail dynamics.
• This approach overcomes limitations of previous methods, providing insights into histone structure beyond crystal structures.
• Findings offer a deeper understanding of the structural basis of the histone code and epigenetic regulation.