Structural Characterization of Human Histone H4.1 by Tandem Nonlinear and Linear Ion Mobility Spectrometry Complemented with Molecular Dynamics Simulations.

Extracellular histone H4 is an attractive drug target owing to its roles in organ failure in sepsis and other diseases. To identify inhibitors using in silico methods, information on histone H4 structural dynamics and three-dimensional (3D) structural coordinates is required. Here, DNA-free histone H4 type 1 (H4.1) was characterized by utilizing tandem nonlinear and linear ion mobility spectrometry (FAIMS-TIMS) coupled to mass spectrometry (MS) complemented with molecular dynamics (MD) simulations. The gas-phase structures of H4.1 are dependent on the starting solution conditions, evidenced by differences in charge state distributions, mobility distributions, and collision-induced unfolding (CIU) pathways. The experimental results show that H4.1 adopts diverse conformational types from compact (C) to partially folded (P) and subsequently elongated (E) structures. Molecular dynamics simulations provided candidate structures for the histone H4.1 monomer in solution and for the gas-phase structures observed using FAIMS-IMS-TOF MS as a function of the charge state and mobility distribution. A combination of the FAIMS-TIMS experimental results with theoretical dipole calculations reveals the important role of charge distribution in the dipole alignment of H4.1 elongated structures at high electric fields. A comparison of the secondary and primary structures of DNA-free H2A.1 and H4.1 is made based on the experimental IMS-MS and MD findings.