Nucleosomes are dynamic entities that are repositioned along DNA by chromatin remodeling processes. A nucleosome repositioned by the switch-sucrose nonfermentable (SWI/SNF) remodeler collides with a neighbor and forms the intermediate "overlapping dinucleosome." Here, we report the crystal structure of the overlapping dinucleosome, in which two nucleosomes are associated, at 3.14-angstrom resolution. In the overlapping dinucleosome structure, the unusual "hexasome" nucleosome, composed of the histone hexamer lacking one H2A-H2B dimer from the conventional histone octamer, contacts the canonical "octasome" nucleosome, and they intimately associate. Consequently, about 250 base pairs of DNA are left-handedly wrapped in three turns, without a linker DNA segment between the hexasome and octasome moieties. The overlapping dinucleosome structure may provide important information to understand how nucleosome repositioning occurs during the chromatin remodeling process.

Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA.

It is now recognized that intrinsically disordered proteins (IDPs) play important roles as hubs in intracellular networks, and their structural characterisation is of significance. However, due to their highly dynamic features, it is challenging to investigate the structures of IDPs solely by conventional methods. In the present study, we demonstrate a novel method to characterise protein complexes using electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) in combination with small-angle X-ray scattering (SAXS). This method enables structural characterisation even of proteins that have difficulties in crystallisation. With this method, we have characterised the Schizosaccharomyces pombe Swi5-Sfr1 complex, which is expected to have a long disordered region at the N-terminal portion of Sfr1. ESI-IM-MS analysis of the Swi5-Sfr1 complex revealed that its experimental collision cross-section (CCS) had a wide distribution, and the CCS values of the most dominant ions were ∼56% of the theoretically calculated value based on the SAXS low-resolution model, suggesting a significant size reduction in the gas phase. The present study demonstrates that the newly developed method for calculation of the theoretical CCSs of the SAXS low-resolution models of proteins allows accurate evaluation of the experimental CCS values of IDPs provided by ESI-IM-MS by comparing with the low-resolution solution structures. Furthermore, it was revealed that the combination of ESI-IM-MS and SAXS is a promising method for structural characterisation of protein complexes that are unable to crystallise.

It has been suggested that the hexasome, in which one of the H2A/H2B dimers is depleted from the canonical nucleosome core particle (NCP), is an essential intermediate during NCP assembly and disassembly, but little structural evidence of this exists. In this study, reconstituted products in a conventional NCP preparation were analyzed by native electrospray ionization mass spectrometry, and it was found that the hexasome, which migrated in a manner almost identical to that of the octasome NCP in native polyacrylamide gel electrophoresis, was produced simultaneously with the octasome NCP. This result might contribute to understanding the assembly and disassembly mechanism of NCPs.

The minimum structural unit of chromatin is the nucleosome core particle (NCP), consisting of 146 bp of DNA wrapped around a histone octamer, which itself contains two H2A/H2B dimers and one (H3/H4)2 tetramer. These multimers possess functionally important tail regions that are intrinsically disordered. In order to elucidate the mechanisms behind NCP assembly and disassembly processes, which are highly related to gene expression, structural characterization of the H2A/H2B dimer and (H3/H4)2 tetramer will be of importance. In the present study, human histone multimers with disordered tail regions were characterized by electrospray ionization (ESI) ion mobility-mass spectrometry (IM-MS) and molecular dynamics (MD) simulation. Experimentally obtained arrival times of these histone multimer ions showed rather wide distributions, implying that multiple conformers exist for each histone multimer in the gas phase. To examine their structures, MD simulations of the histone multimers were performed first in solution and then in vacuo at four temperatures, resulting in a variety of histone multimer structures. Theoretical collision cross-section (CCS) values calculated for the simulated structures revealed that structural models with smaller CCS values had more compact tail regions than those with larger CCS values. This implied that variation of the CCS values of the histone multimers were primarily due to the random behaviors of the tail regions in the gas phase. The combination of IM-MS and MD simulation enabled clear and comprehensive characterization of the gas-phase structures of histone multimers containing disordered tails.

Self-propelled droplets can spontaneously move using chemical energy. In several reports of self-propelled droplets, interfacial chemical reactions occur at the oil/aqueous interface to induce the Marangoni flow. While the dynamics of interfacial tension is essential to the droplet motion, there are few reports that quantitatively discuss the moving mechanism based on interfacial tension measurements. In this study, we focused on the selfpropelled motion of an aqueous droplet in the oil phase, where the surfactant monoolein reacts with bromine at the interface, and estimated the physicochemical parameters related to the droplet motion based on the time series of interfacial tension. These results may reveal the general mechanism for the self-propelled motion of aqueous/oil droplets driven by the interfacial chemical reaction.

The histone H2A/H2B dimer is a component of nucleosome core particles (NCPs). The structure of the dimer at the atomic level has not yet been revealed. A possible reason for this is that the dimer has three intrinsically disordered tail regions: the N- and C-termini of H2A and the N-terminus of H2B. To investigate the role of the tail regions of the H2A/H2B dimer structure, we characterized behaviors of the H2A/H2B mutant dimers, in which these functionally important disordered regions were depleted, using mass spectrometry (MS). After verifying that the acetylation of Lys residues in the tail regions had little effect on the gas-phase conformations of the wild-type dimer, we prepared two histone H2A/H2B dimer mutants: an H2A/H2B dimer depleted of both N-termini (dN-H2A/dN-H2B) and a dimer with the N- and C-termini of H2A and the N-terminus of H2B depleted (dNC-H2A/dN-H2B). We analyzed these mutants using ion mobility-mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS). With IM-MS, reduced structural diversity was observed for each of the tail-truncated H2A/H2B mutants. In addition, global HDX-MS proved that the dimer mutant dNC-H2A/dN-H2B was susceptible to deuteration, suggesting that its structure in solution was somewhat loosened. A partial relaxation of the mutant's structure was demonstrated also by IM-MS. In this study, we characterized the relationship between the tail lengths and the conformations of the H2A/H2B dimer in solution and gas phases, and demonstrated, using mass spectrometry, that disordered tail regions play an important role in stabilizing the conformation of the core region of the dimer in both phases.

Histone tails, which protrude from nucleosome core particles (NCPs), play crucial roles in the regulation of DNA transcription, replication, and repair. In this study, structural diversity of nucleosomes was investigated in detail by analyzing the observed charge states of nucleosomes reconstituted with various lengths of DNA using positive-mode electrospray ionization mass spectrometry (ESI-MS) and molecular dynamics (MD) simulation. Here, we show that canonical NCPs, having 147 bp DNA closely wrapped around a histone octamer, can be classified into three groups by charge state, with the least-charged group being more populated than the highly charged and intermediate groups. Ions with low charge showed small collision cross sections (CCSs), suggesting that the histone tails are generally compact in the gas phase, whereas the minor populations with higher charges appeared to have more loosened structure. Overlapping dinucleosomes, which contain 14 histone proteins closely packed with 250 bp DNA, showed similar characteristics. In contrast, mononucleosomes reconstituted with a histone octamer and longer DNA (≥250 bp), which have DNA regions uninvolved in the core-structure formation, showed only low-charge ions. This was also true for dinucleosomes with free DNA regions. These results suggest that free DNA regions affect the nucleosome structures. To investigate the possible structures of NCP observed in ESI-MS, computational structural calculations in solution and in vacuo were performed. They suggested that conformers with large CCS values have slightly loosened structure with extended tail regions, which might relate to the biological function of histone tails.