Search

The canonical nucleosome, which represents the major packaging unit of eukaryotic chromatin, has an octameric core composed of two histone H2A-H2B and H3-H4 dimers with ∼147 base pairs (bp) of DNA wrapped around it. Non-nucleosomal particles with alternative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Using cryo–electron microscopy, we solved the structure of the H3-H4 octasome, a nucleosome-like particle with a di-tetrameric core consisting exclusively of the H3 and H4 histones. The core is wrapped by ∼120 bp of DNA in 1.5 negative superhelical turns, forming two stacked disks that are connected by a H4-H4’ four-helix bundle. Three conformations corresponding to alternative interdisk angles were observed, indicating the flexibility of the H3-H4 octasome structure. In vivo crosslinking experiments detected histone–histone interactions consistent with the H3-H4 octasome model, suggesting that H3-H4 octasomes or related structural features exist in cells.

Native mass spectrometry (MS) is an analytical technique used to determine the molecular mass of protein complexes without cross-linking. Size exclusion chromatography (SEC) coupled with native MS using conventional electrospray ionization (ESI) has been reported to allow online buffer exchange. To detect a wide variety of protein complexes without a collapse in the ionization process, it is important to build an online system that enables robust analysis with a low flow rate.We created an online native MS system equipped with nanoESI connected to the SEC component (online SEC/nanoESI system) and optimized several parameters for SEC separation and ionization. The constructed system was used to measure a solution consisting of a protein mixture of various molecular masses (10-300 kDa) to verify characteristics such as the measurable molecular mass range, reproducibility, and online buffer exchange.The optimal flow rates for SEC separation and nanoESI analysis using this system were 200 and 1 μL/min, respectively. This system was able to analyze proteins in the ranges of 10-300 and 20-300 kDa for protein samples in ammonium acetate and nonvolatile buffer, respectively. Furthermore, the results of consecutive measurements showed that the relative standard deviations of the retention times and observed masses for each protein were sufficiently small.We created an online SEC/nanoESI system and evaluated its utility for the analysis of various proteins in conventional measurement solvent and nonvolatile buffer. As a result, the structural stability and resolution of the proteins were found to be sufficient when using online buffer exchange. Therefore, this online SEC/nanoESI system would be a useful technique for obtaining mass spectra of various proteins automatically with good resolution, simply by loading samples into an autosampler.

Small molecules with polar functional groups, including substituted phenethylamines, are commonly analyzed by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). Analyte molecules are mostly detected in protonated and cation-adducted forms through positive-ion electrospray ionization-mass spectrometry (ESI-MS). However, the ESI of substituted phenethylamines commonly provides an intense signal of fragment ions by ESI in-source collision-induced dissociation (IS-CID), which hinders the unambiguous identification of phenethylamines. This phenomenon was approximated as a unimolecular dissociation model, and the dissociation efficiency was evaluated by various quantum chemistry calculations to determine the ESI IS-CID efficiency. The calculated results were consistent with the experimental data, when the dissociation threshold energy of phenethylamines was calculated using the post-Hartree-Fock (post-HF) method, CCSD(t)/cc-pVTZ//MP2(full)/6-311++G(d,p). In contrast to post-HF methods, the utilization of density functional theory calculations with a suitable functional is recognized as an accurate and competitive low-cost approach. In particular, ωB97-XD, M06-2X-D3, and recently developed Minnesota functionals, such as M11, MN12-SX, and MN15, provided reliable results, as in the case of the post-HF method. The results obtained by the recently developed double hybrid functionals, DSD-PEBP86-D3(BJ), PBE0-DH, and PBE-QIDH, were also reliable. The consideration of ESI IS-CID can facilitate the identification of analyte molecules because most phenethylamines, except for N-methylated analogs, provide an intense signal in the ESI mass spectrum.

The canonical nucleosome, which represents the predominant packaging unit in eukaryotic chromatin, has an octameric core made up of two histone H2A-H2B and H3-H4 dimers with ~147 base-pair (bp) DNA wrapping around it. Non-nucleosome particles with alterative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Here we solved the structure of the H3-H4 octasome, which is a nucleosome-like particle with a core made up of four H3-H4 dimers. Two conformations, open and closed, are determined at 3.9 Å and 3.6 Å resolutions by cryo-electron microscopy, respectively. The H3-H4 octasome, made up of a di-tetrameric core, is wrapped by ~120 bp DNA in 1.5 negative superhelical turns. The symmetrical halves are connected by a unique H4-H4’ interface along the dyad axis. In vivo crosslinking of cysteine probes placed at another unique H3-H3’ interface demonstrated the existence of the H3-H4 octasome in cells.

Inorganic salts and nonvolatile-buffer components affect the structure and stability of proteins, and some protein complexes are unable to maintain their function and structure without them. However, it is well-known that these components cause suppression of analyte ionization during the electrospray ionization process. Thus, to establish appropriate methods for observation of the intact ions of protein and DNA complexes by native mass spectrometry (native MS) in the presence of nonvolatile buffer components, we herein examined the effect of ammonium acetate addition to a model homotetramer protein, alcohol dehydrogenase (ADH), which was prepared in a range of nonvolatile buffers, including Tris-HCl, phosphate, and HEPES buffers. Furthermore, native MS of nucleosome core particle (NCP), a large protein-DNA complex, prepared in nonvolatile buffer, was also examined. Intact ADH and NCP ions could be observed upon the addition of ammonium acetate, but NCP does not require as high of a concentration of ammonium acetate as ADH. Well-resolved peaks with different charge numbers could be observed for NCP prepared in Tris-HCl by addition of a lower amount of ammonium acetate than for ADH. This suggests that the effects of additives on native MS of biomolecular complexes can vary depending on the intramolecular interactions present. More specifically, NCP is stabilized mainly by electrostatic interactions, whereas the ADH tetramer depends on the presence of hydrophobic interactions between the four subunits. The results presented herein therefore are expected to contribute to structural biology studies of unstable protein-DNA complexes that are formed transiently during the transcription 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.

2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and

Post-translational modifications (PTMs) of histone N-terminal tails in nucleosome core particle (NCP), such as acetylation, play crucial roles in regulating gene expression. To unveil the regulation mechanism, atomic-level structural analysis of in-vitro modified NCP is effective with verifying the PTMs of histones. So far, identification of PTMs of NCP originating from living cells has mainly been performed using mass spectrometry (MS) techniques, such as bottom-up approach. The bottom-up approach is the most established method for protein characterization, but it does not always provide sufficient information on the acetylated sites of lysine residues in the histone tails if trypsin digestion is carried out. For histone proteins, which have many basic amino acids, trypsin generates too many short fragments that cannot be perfectly analyzed by tandem MS. In this study, we investigated the in vitro acetylation sites in the histone H3 tail using a top-down sequence analysis, matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiment, in combination with aminopeptidase digestion. Aminopeptidase can cleave peptide bonds one-by-one from the N-terminus of peptides or proteins, generating N-terminally truncated peptides and/or proteins. As a result, it was identified that this method enables sequence characterization of the entire region of the H3 tail. Also, application of this method to H3 in in-vitro acetylated NCP enabled assigning acetylation sites of H3. Thus, this method was found to be effective for obtaining information on in-vitro acetylation of NCP for structural biology study.

A convenient analytical system for protein-ligand interactions under crude conditions was developed using native mass spectrometry (MS). As a model protein, Escherichia coli (E. coli) dihydrofolate reductase (DHFR) with and without a histidine tag was used for the study. First, overexpressed DHFR with a His-tag was roughly purified with a Ni-sepharose resin and subjected to native mass spectrometry with or without incubation with an inhibitor, Methotrexate (MTX). Even only with the minimum cleanup by the Ni-sepharose resin, intact ions of DHFR-nicotinamide adenine dinucleotide phosphate (NADPH) and DHFR-NADPH-ligand complexes were successfully observed. By optimizing the preparation procedures of the crude sample for native MS, e.g., avoiding sonication for cell lysis, we successfully observed intact ions of the specific DHFR-NADPH-MTX ternary complex starting with cultivation of E. coli in ≤ 25 mL medium. When the crude DHFR sample was mixed with two, four, or eight candidate compounds, only ions of the specific protein-ligand complex were observed. This indicates that the present system can be used as a rapid and convenient method for the rough determination of binding of specific ligands to the target protein without the time-consuming purification of protein samples. Moreover, it is important to rapidly determine specific interactions with target proteins under conditions similar to those in "real" biological systems. Graphical abstract.

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.

Nucleosomes in contact In eukaryotic cells, genomic DNA must be compacted to fit inside the nucleus. A key player in DNA packaging is the nucleosome, which comprises a segment of DNA wrapped around an octamer of histone proteins. During replication and transcription, nucleosomes must reposition themselves on the DNA. In this process, nucleosomes can collide to form a dinucleosome. Kato et al. report a high-resolution crystal structure of a dinucleosome. One of the octamers has lost a histone dimer so that the dinucleosome comprises an octamer and a hexamer. The structure may represent an intermediate during chromatin remodeling. Science , this issue p. 205

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 self-propelled 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.

It is well known that various modifications of histone tails play important roles in the regulation of transcription initiation. In this study, some lysine (Lys) and arginine (Arg) residues were acetylated and deiminated, respectively, in the histone H2A/H2B dimer, and charge-neutralization effects on the dimer structure were studied by native mass spectrometry. Given that both acetylation and deimination neutralize the positive charges of basic amino acid residues, it had been expected that these modifications would correspondingly affect the gas-phase behavior of the histone H2A/H2B dimer. Contrary to this expectation, it was found that Arg deimination led to greater difficulty of dissociation of the dimer by gas-phase collision, whereas acetylation of Lys residues did not cause such a drastic change in the dimer stability. In contrast, ion mobility-mass spectrometry (IM-MS) experiments showed that arrival times in the mobility cell both of acetylated and of deiminated dimer ions changed little from those of the unmodified dimer ions, indicating that the sizes of the dimer ions did not change by modification. Charge neutralization of Arg, basicity of which is higher than Lys, might have triggered some alteration of the dimer structure that cannot be found in IM-MS but can be detected by collision in the gas phase.

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.

Ion mobility mass spectrometry was employed to study the structure of the βB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of βB3 crystallin across residues 115–126 and 152–166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.

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.

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.

The topology and dynamics of melittin within the liposome were investigated by a mass spectrometry coupled with acetylation. The MALDI-TOF MS and MALDI-QIT-TOF MS/MS analyses revealed that only N-terminal amine of melittin was dominantly acetylated in the presence of liposome although all of four primary amines were completely and rapidly acetylated in aqueous solution. This result indicates that melittin adopts the N-terminal-outside transmembrane topology within the liposome. The time course of acetylation followed the first-order kinetics at any examined temperatures (6–30 °C). The rate constant was less than that of the acetylation of melittin in aqueous solution. The activation energy for acetylation (74 kJ mol −1 ) was comparable to that for dissociation of a lipid monomer from the membrane, suggesting a float-like longitudinal motion of melittin within the liposome. These results demonstrate that a mass spectrometry combined with chemical modification is very efficient way for clarifying the topology and dynamics of peptides bound to the membrane.

We investigated the topology and dynamics of melittin within the liposomes using mass spectrometry combined with acetylation. According to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry/mass spectrometry (MALDI-QIT-TOF MS/MS) analyses, melittin within the liposomes was mono acetylated; with the acetylated position being the N-terminal of melittin. This acetylation followed first-order kinetics. The rate constant was less than that of the acetylation of melittin in aqueous solution. Thus, it is suggested that the observed rate constant is that for the release of the N-terminal region of melittin to a water phase from the hydrophobic core of the liposomes. This approach has opened a new method in the study of dynamics of transmembrane helices within the membranes using mass spectrometry.

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 nucleosome core particle (NCP) is the minimum structural unit of chromatin and is composed of a histone octamer and 146 base pairs of DNA. The DNA is wrapped around the histone octamer, which consists of two H2A/H2B dimers and one (H3/H4)2 tetramer. These histone multimers contain intrinsically disordered tail regions that are functionally important for NCP assembly and disassembly, which are highly relevant to gene expression. In order to elucidate the mechanisms underlying these processes, it is of importance to characterize structures of the H2A/H2B dimer and (H3/H4)2 tetramer. In the present study, we investigated gas-phase structures of these two histone multimers having disordered tail regions using electrospray ionization ion mobility-mass spectrometry (IM-MS) and molecular dynamics (MD) simulation. IM-MS experiments of the histone multimers showed that their arrival-time distributions were rather wide, implying that each histone multimer could have multiple conformers 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, providing a variety of their gas-phase structures. By calculating theoretical collision cross-section (CCS) values of these structures, it was found that histone multimer structures with smaller CCS values had more compact tail regions than those with larger CCS values. This finding suggests that multiple conformers of the histone multimers are primarily due to the random behaviors of the tail regions in the gas phase. Thus, the combination use of IM-MS and MD simulation enables us to clearly characterize gas-phase structures of proteins containing disordered tails.