Gas-Phase Structure of the Histone Multimers Characterized by Ion

Mar 13, 2013 - This implied that variation of the CCS values of the histone multimers were primarily due to the random behaviors of the tail regions i...
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Gas-Phase Structure of the Histone Multimers Characterized by Ion Mobility Mass Spectrometry and Molecular Dynamics Simulation Kazumi Saikusa,† Sotaro Fuchigami,† Kyohei Takahashi,†,§ Yuuki Asano,†,∥ Aritaka Nagadoi,† Hiroaki Tachiwana,‡ Hitoshi Kurumizaka,‡ Mitsunori Ikeguchi,† Yoshifumi Nishimura,*,† and Satoko Akashi*,† †

Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan ‡ Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan S Supporting Information *

ABSTRACT: 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 gasphase structures of histone multimers containing disordered tails. intrinsic flexibility.1,8 Also, no structure has yet been reported for the H2A/H2B dimer or (H3/H4)2 tetramer in isolation. It is now recognized that proteins with unstructured regions such as histone proteins, called intrinsically disordered proteins (IDPs), play important roles as hubs in intracellular networks of protein interactions, and as such, their structural characterization is of great importance for understanding eukaryotic cellular function. However, approaches to effectively elucidate IDP structure are still in an early stage of investigation. In general, IDPs cannot crystallize, and so their structures have been studied by other methods such as NMR spectroscopy, small-angle X-ray scattering (SAXS), mass spectrometry (MS), and so on.9−14 NMR can provide structural information for proteins in solution, even of IDPs, but this technique encounters difficulty when applied to the analysis of huge

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iological functions in living cells are highly regulated by a variety of protein machineries. Nucleosome core particles (NCPs) are one of such protein machinery, and the dynamic processes governing their assembly and disassembly are essential for gene expression. NCPs are the basic structural unit of eukaryotic chromatin, with each consisting of approximately 146 base pairs of DNA wrapped around a histone octamer that is composed of two H2A/H2B dimers and a (H3/H4)2 tetramer.1 It has been suggested that various posttranslational modifications on the N-terminal flexible tails of histone proteins induce dynamic structural changes in NCPs and promote regulatory activity on DNA transcription, replication, and repair.2−7 Thus, characterizing the structures of the histone H2A/H2B dimer and (H3/H4)2 tetramer and their disordered tail regions will be of importance in understanding the mechanisms behind NCP assembly and disassembly. So far, the structures of the NCP and histone octamers (H2A/H2B/H3/H4)2 have been determined at the atomic level by X-ray crystallography, but the disordered tail regions remain absent in the X-ray structures due to their high © 2013 American Chemical Society

Received: February 5, 2013 Accepted: March 13, 2013 Published: March 13, 2013 4165

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Figure 1. (A) ESI mass spectrum of the H2A/H2B dimer in 2 M ammonium acetate obtained in the TOF mode. (B) ESI-IM-MS data of the H2A/ H2B dimer. (a) ESI mass spectrum obtained in the IM-MS mode. Multiply charged ions not only of the H2A/H2B dimer (red arrows) but also of the H2A monomer (blue arrows) and H2B monomer (green arrows) were observed. (b) Arrival-time distribution of 11+ ion of the dimer. (c) Ion mobility contour plot of the dimer, in which the m/z values (x-axis) were plotted vs the arrival times (y-axis, ms). Relative abundances of the ions are indicated by a color gradation in the inset. Dashed lines indicate the existence of two populations for 10+ to 13+ charged ions of the dimer.

tail regions using electrospray ionization (ESI) IM-MS and MD simulation. The two histone multimers were prepared by refolding of recombinant histone proteins; the multimers were then subjected to MS to examine their stabilities. Then, IM-MS was utilized to characterize the multimer conformations based on their CCS distributions. We also performed 2-step all-atom MD simulations of the histone multimers: first in explicit aqueous solution to confirm the flexibilities of histone tails, then in vacuo at four different temperatures to reveal atomiclevel structures of histone multimers in the gas phase and to estimate the distribution of their CCS values. Finally, the experimental CCS distributions were compared with those obtained by MD simulations to gain potential atomic-level structures of the histone multimers in the gas phase as well as the behaviors of their disordered tail regions.

protein complexes. The use of SAXS enables coarse-grained models of any protein or protein complex to be determined, although it is challenging to analyze proteins that can adopt multiple conformations. MS is also a favorable tool for analyzing protein complexes that cannot crystallize because components of a mixture of protein complexes can be separated according to mass.15,16 Furthermore, ion mobility-mass spectrometry (IM-MS)17 can provide not only masses for protein complexes but also their collision cross-section (CCS) values, which yield information on their compactness and topology. Therefore, IM-MS has recently come to be recognized as an effective technique for characterizing the structures of protein complexes in native forms.18−20 It has also been demonstrated that combining IM-MS with other methods, such as molecular dynamics (MD) simulation21−27 and SAXS,28 provides for more effective analysis. For IDPs in particular, structural characterization using this combination of IM-MS and MD simulation is a promising approach because MD simulation can suggest possible candidate structures for IDPs at atomic resolution. Indeed, atomic-level structures of amyloid β-protein24−26 and p5327 have already been successfully reported using this integrated approach. Thus, an analysis that employs IM-MS and MD simulation would be suitable for revealing the structures of histone multimers. In the present study, we investigated gas-phase structures of H2A/H2B dimers and (H3/H4)2 tetramers having disordered

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EXPERIMENTAL SECTION Experimental outline is indicated below. Detailed experimental procedures are described in the Supporting Information, Supplementary Materials and Methods section. Preparation of Histone Proteins for ESI-MS. Histone multimers were prepared by refolding the recombinant human H2A, H2B, H3, and H4 monomers (Figure S-1 in the Supporting Information) according to techniques outlined in previous reports.29,30 4166

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Figure 2. (A) ESI mass spectrum of the (H3/H4)2 tetramer in 4 M ammonium acetate obtained in the TOF mode. (B) ESI-IM-MS data of the (H3/H4)2 tetramer. (a) ESI mass spectrum obtained in the IM-MS mode. Multiply charged ions not only of the (H3/H4)2 tetramer (red arrows) but also of the H3/H4 dimer (blue arrows) were observed. (b) Arrival-time distribution of 16+ of the tetramer. (c) Ion mobility contour plot of the tetramer. Relative abundances of the ions are indicated by a color gradation in the inset. Dashed lines indicate the existence of two populations for 14+ to 17+ charged ions of the tetramer.

ESI-IM-MS of Histone Multimers. Mass spectra and arrival times for the histone multimers were acquired by Triwave SYNAPT G2 HDMS (Waters, Milford, MA) with a nanoESI source.28,31−33 The samples in ammonium acetate solutions (pH 6.8) were deposited in gold-coated borosilicate capillaries and placed in the nanoESI source. To avoid artificial activation of the analyte ions, the measurement conditions for ESI-MS and ESI-IM-MS were optimized so as to be as mild as possible. Two dimensional (2D) contour plots of arrival time versus m/z were generated using Drift Scope software (Waters). Because arrival times obtained by IM-MS contained mass-dependent flight times between the ion mobility cell and the time-of-flight (TOF) analyzer, they were converted into CCS values using the equation given by Ruotolo et al.34 as indicated in the Supporting Information. MD Simulations and Analysis. All-atom MD simulations were performed for the histone multimers first in solution then in vacuo. To prepare the initial structures for simulation, the histone multimer in X-ray structure of human NCP (PDB ID: 3AFA)35 was used as an original model. Tails of histone proteins, which were invisible in the X-ray structure, were built with the PyMOL software.36 Then, the initial structures of the H2A/H2B dimer and (H3/H4)2 tetramer were solvated in cubic boxes of high-salt solutions of 2 and 4 M NaCl, respectively. MD simulations of the histone multimers in an explicit aqueous solution were performed for 10 ns using the MD program MARBLE.37 Subsequently, 10 structures obtained from snapshots (taken every 1 ns) of the MD simulations in

solution were subjected to gas-phase MD simulation. The charged sites were fixed by a Monte Carlo sampling method with the Metropolis algorithm38,39 so as to minimize the charge repulsion. Following equilibration of the initial structures with 11+ (H2A/H2B dimer) or 16+ ((H3/H4)2 tetramer) charges, gas-phase simulations were carried out for 5 ns at four different temperatures (T = 100, 200, 300, and 400 K). Each simulation was performed 15 times with randomly generated initial velocities. Using the final snapshots of the simulations, we obtained structure ensembles of the histone multimers in vacuo that consisted of 150 structures (10 initial structures × 15 trials) at each of the four different temperatures.

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RESULTS AND DISCUSSION ESI-MS of Histone Multimers. Previous research has found that when the H2A/H2B dimer is prepared in a solution containing an extremely high concentration of ammonium acetate, the dimer ions can be successfully observed in the ESI mass spectra without dissociation into the component monomers.29 Similarly, in the case of the (H3/H4)2 tetramer, we successfully observed intense tetramer ions by ESI-MS when prepared in 4 M ammonium acetate, as shown in Figure S-2 in the Supporting Information. These observations support the conclusion that the histone multimer ions could survive in the gas phase only when the protein samples were prepared in extremely high concentrations of ammonium acetate. In the present study, ESI-MS experiments were performed for the 4167

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helical regions. This indicated that the helical regions were considerably stable, in marked contrast to the disordered terminal regions. The stability of the helical regions was also confirmed quantitatively by the time course of the root-meansquare displacement (RMSD) of Cα atoms with respect to the corresponding structures of the NCP as shown in Figure S-3A in the Supporting Information. On the other hand, tail regions, the N-terminal regions for all histones and the C-terminal region for H2A, demonstrated disordered characteristics (presented in Figure 3). This was quantified by the rootmean-square fluctuation (RMSF) values larger than 2 Å for the amino acid residues in the tail regions (Figure S-3B in the Supporting Information). Here, the five regions that contained more than five amino acid residues with RMSF values larger than 2 Å were defined as the tail regions: they were residues −3 through 14 (N-tail) and 99 through 129 (C-tail) of H2A, −3 through 34 of H2B, −3 through 43 of H3, and −3 through 23 of H4 (Figure S-1 in the Supporting Information). In contrast, the other regions were defined as the core for each histone multimer. Detailed discussion is described in the Supporting Information, Discussion 1. For the MD simulated structures of the histone multimers in solution, the average charge states of 15.8 (H2A/H2B dimer) and 24.8 ((H3/H4)2 tetramer) were calculated, respectively, using the proposed equation of charge-to-accessible surface area (ASA) correlation (eq 1 in the Supporting Information).42 These calculated values were significantly different from the actually observed charge states of 11.1 and 15.7 for these multimers in 2 or 4 M ammonium acetate in the ESI mass spectra (Figures 1A and 2A). In contrast, average charge states of the core regions in the 10 MD-simulated structures of the H2A/H2B dimer and (H3/H4)2 tetramer (Figure 3) were calculated as 11.4 (H2A/H2B dimer core) and 15.9 ((H3/H4)2 tetramer core), both of which were close to the observed values. Thus, structures of both the H2A/H2B dimer and H3/ H4 tetramer in the gas phase seemed to be contracted much more than those in solution. MD Simulation of Histone Multimers in Vacuo. We subsequently investigated the behavior of the histone multimers in the gas phase. MD simulation in vacuo was performed for the 10 initial structures of each histone multimer as indicated in Figure 3 with 11+ (for the H2A/H2B dimer) and 16+ (for the (H3/H4)2 tetramer) charge states, respectively. These charge states were defined as the “ESI-charge states”, which correspond to the most dominant multimer ions observed in the mass spectra (Figures 1 and 2). Protonated sites in the initial structures were determined so as to minimize the charge repulsion, as shown in Table S-1 in the Supporting Information. Following equilibration of the initial structures in the ESIcharge state for 600 ps at 300 K, gas-phase simulations were performed for 5 ns. CCS values were then calculated for the obtained MD trajectories (Figure S-4A in the Supporting Information). The transition from solution into the gas phase promptly reduced the CCS values of the multimers to about two-thirds of those of the initial structures, and these values remained constant throughout the 5 ns calculation. No dependence on the initial structures was found in the gasphase structures (Figure S-4B in the Supporting Information). To evaluate these MD-simulated structures, the theoretical CCS values of the 150 final MD-simulation structures for the H2A/H2B dimer and (H3/H4)2 tetramer in the ESI-charge state were calculated according to eq 2 in the Supporting Information.34,43,44 Figure 4A(c) shows the CCS histogram of

H2A/H2B dimer in 2 M ammonium acetate and the (H3/H4)2 tetramer in 4 M ammonium acetate (Figures 1A and 2A). In the ESI mass spectra, ions with 10+ through 13+ charges were observed for the H2A/H2B dimer (Figure 1A), whereas the (H3/H4)2 tetramer exhibited 14+ through 17+ charge states (Figure 2A). Each histone multimer exhibited a narrow charge-state distribution, implying a single population with native-like structure in general.40,41 IM-MS of Histone Multimers. Next, IM-MS experiments were performed for the histone multimers, and the CCS values of the observed ions were analyzed. In the mass spectra obtained in the IM-MS mode (Figures 1B(a) and 2B(a)), peaks at low m/z regions were observed; these peaks were not detected in the TOF-mode mass spectra (Figures 1A and 2A). Since the multimer ions were energetically activated a little by the bias voltage at the ion mobility entrance into the pressurized ion mobility region in the IM-MS mode, collision of the multimer ions with the gas molecules was unavoidable to some degree. Consequently, highly charged multimer ions and dissociated monomer ions appeared at lower m/z regions in Figures 1B(a) and 2B(a), although the measurement condition was optimized to be mild. To discuss the structures of the originally observed ions in the ESI mass spectra, we focus here on the relatively low-charged ions observed in the mass spectra in both modes. To analyze the relationship between the m/z values and arrival times of the ions detected in the IM-MS mode, the 2D contour plot for each histone multimer was constructed as shown in Figures 1B(c) and 2B(c). The 2D contour plots revealed the existence of two structural populations, a compact (Group I) and a loose (Group II) one, for each H2A/H2B dimer and (H3/H4)2 tetramer. These two populations are also visible in the arrival-time distribution plots of the most dominantly observed ions of the multimers (11+ for H2A/H2B, 16+ for (H3/H4)2) (Figure 1B(b) and 2B(b)). Their structural populations were then investigated by MD simulation in detail. MD Simulation of Histone Multimers in Solution. In order to analyze the structures of the histone multimers observed through IM-MS, consecutive MD simulations in solution and in vacuo were performed. At first, MD simulations in solutions containing 2 M NaCl (for the H2A/H2B dimer) or 4 M NaCl (for the (H3/H4)2 tetramer) were run for 10 ns. Figure 3 shows 10 structures of the histone multimers, each taken 1 ns apart in the MD trajectories, superimposed using the

Figure 3. Structural and dynamic properties of the histone multimers in solution. A total of 10 structures for each histone multimer are superimposed with the helical regions of the histone multimers aligned; H2A is shown in yellow, H2B in red, H3 (chains A and E) in blue and cyan, and H4 (chains B and F) in green and lime-green. 4168

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summarized in the forms of CCS histograms (Figure 4) and cumulative frequency distributions (Figure S-5A in the Supporting Information). MD simulated structures of the H2A/H2B dimer at 400 K mirrored the CCS distribution at 300 K, with a similar center of 2260 Å2. On the other hand, the distributions at 200 and 100 K showed slightly larger center values than at 300 K. As indicated in Figure 4A and Figure S-5A(a) in the Supporting Information, the distribution envelope at each temperature slightly shifted toward high CCS values as the simulation temperature decreased. In the experimental CCS distribution of the 11+ charged ion of the H2A/H2B dimer, two peaks were distinguishable, suggesting the existence of two populations: Group I with a center at 2270 Å2 and Group II at 2480 Å2 in the CCS distribution (Figure 4A(a)). Group I might correspond to the simulated model at high temperatures (300−400 K) (Figure 4A(b),A(c)), whereas the CCS envelope of the simulated models at low temperature (100 K) (Figure 4A(e)) might represent Group II. The CCS histogram of the simulated models of the (H3/ H4)2 tetramer exhibited a significant shift to high CCS values as the temperature decreased (Figure 4B(b)−(e)). The distribution envelope for the models at 400 K showed the smallest center value at 3490 Å2, while that for the models at 100 K presented a wider distribution having the largest center value at 3960 Å2. In the experimental CCS distribution of the 16+ ion of the tetramer, as shown in Figure 4B(a), two populations, Groups I and II, were recognized, with center values of 3580 Å2 and 3810 Å2, respectively. By analyzing the observed CCS values of the simulated structures at each temperature set, it appeared that Groups I and II corresponded to the simulated models at high (300−400 K) and low (100−200 K) temperatures, respectively. As another factor contributing to the structural variation observed in ESI-IM-MS, charge sites were considered and subjected to a follow-up inquiry. However, no effect from random alteration of the charged sites was recognized, as demonstrated in Figure S-5B in the Supporting Information. Structural Properties of the Histone Multimers in the Gas Phase. As mentioned above, the gas-phase MD simulation experiments at various temperatures provided compelling evidence for the production of structural variety for each histone multimer observed in IM-MS. Figure 5 shows the structures of the histone multimers with the smallest and largest CCS values among 600 structural variants obtained by gas-

Figure 4. Histograms of the CCS distributions for the MD simulated structures of the H2A/H2B dimer (A) and (H3/H4)2 tetramer (B). The experimental CCS distributions of the multimer ions in the ESIcharge state (11+ for the H2A/H2B dimer and 16+ for the (H3/H4)2 tetramer) are indicated in the top panel (a). Two structural populations were recognized in the experimental CCS distributions. The full widths at half-maximum are shown in light blue and green for the respective population. The center CCS value corresponding to each population is indicated above the line. The CCS values of the MD simulated structures in the ESI-charge state in the gas phase at 400 K (b), 300 K (c), 200 K (d), and 100 K (e) are indicated in the forms of histograms. Each bar therein has a width of 50 Å2 for the H2A/H2B dimer or 100 Å2 for the (H3/H4)2 tetramer.

the MD-simulated structure for the H2A/H2B dimer at 300 K: it can be seen that its distribution spans from 2150 to 2400 Å2 and is centered at 2280 Å2. The CCS histogram for the (H3/ H4)2 tetramer at 300 K (Figure 4B(c)) exhibited a distribution from 3400 to 4000 Å2 with a center of 3660 Å2. Subsequent analysis clearly suggested that the MD-simulated structures of both the H2A/H2B dimers and (H3/H4)2 tetramers at 300 K possess a single population profile with a relatively narrow CCS distribution, this is inconsistent with the experimental results obtained by IM-MS (Figure 4A(a),B(a)). To investigate the reason for this discrepancy, some more MD-simulation sets were performed at varying temperatures. This is because solvent evaporation during ESI processes triggers cooling of the analyte protein ions;45,46 acting in opposition, the analyte protein ions collide with the neutral gas in the ion mobility cell, which may activate the ions by collisional heating.47,48 Here, to examine temperature effect, the simulation was carried out for 5 ns at 100K, 200 K, and 400 K in the same manner as the previous 300 K trials. The results are

Figure 5. Structures of the H2A/H2B dimer and (H3/H4)2 tetramer with the smallest (A, C) and largest (B, D) CCS values as obtained by MD simulation. H2A is indicated in yellow, H2B in red, H3 (chain A and E) in blue and cyan, H4 (chain B and F) in green and lime-green. Tail regions are indicated in purple. A surface representation is overlaid with each ribbon model. 4169

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of charge numbers and dielectric constants, it would be possible to provide more accurate and detailed structural information for IDPs based on the results of ESI-IM-MS. It would also be significant to analyze the structural change of the multimers by MD simulation with altered charge states and charged sites at various temperatures. The lack of the migration of protons within the multimer ions during MD simulation and the extremely shorter simulation length (5 ns) compared with the actual IM-MS time scale might have caused generation of only a single structural population, although the IM-MS experiments suggested two structural populations. These issues remain to be revealed in the future to clarify what happens in assemblies of proteins with disordered regions during ESI-IM-MS analysis.

phase MD simulation at four temperatures. The structures of histone multimers with the smallest CCS values (Figure 5A,C), which originated from MD simulation at 400 K, contained more compressed tail regions than those with the largest CCS values (Figure 5B,D), which were obtained from MD simulation at 100 K. In the case of the (H3/H4)2 tetramer, which has a cleft between two H3/H4 dimers that is retained in the initial structure we used for in vacuo calculations (Figure 3B), it appeared that the cleft sizes varied to some extent in the two simulated structures (Figure 5C,D); however, the size of the cleft did not vary as much as that of the tail regions. Therefore, it seems that the flexible tail regions are the main contributors to the variety of structures and sizes observed in the gas phase. To investigate in detail the influence of the tail and core regions on the observed structural variety, hypothetical multimers with zero or one flexible tails were considered: four types of multimers for H2A/H2B dimer (each having no tails, a H2A N-tail, a H2A C-tail, and a H2B Ntail) and three types of multimers for (H3/H4)2 tetramer (each having no tails, a H3 N-tail, and a H4 N-tail) and were examined as described in the Supporting Information (Table S2, Figure S-6, and Discussion 2). Then, it was verified that tail length appears to be a major factor affecting the structural variety of histone multimers in the gas phase. Analysis of the Gas-Phase Structures of Proteins with Disordered Regions. Hitherto, some IDP structures in the gas phase have been investigated by a joint approach combining IM-MS with MD simulation; Bowers and co-workers characterized a completely disordered, small protein, amyloid beta-protein (Aβ42),24,25 and Pagel et al. reported the gasphase structure of p53, a protein with folded domains connected by disordered regions.27 These studies showed typical gas-phase structures for the proteins obtained by MD simulation, suggesting that structural changes in the disordered regions while in the gas phase were primarily responsible for the size reduction of the protein ions. In the present study, IMMS of the histone multimers clearly showed their structural variety in the gas phase. In aqueous solutions with high salt concentrations, hydrophobic interactions are greatly strengthened and stabilize core structures of the multimers, whereas the tail regions fluctuate randomly and widely, as demonstrated by MD simulation. In the gas phase, however, MD simulation suggested that the variety of structures was likely due to varying degrees of collapse of the flexible tail regions, which would have been induced by complex dehydration in the ESI process. As described above, in the analysis of the gas-phase structures of histone multimers, temperature was found to be an important parameter in MD simulation in order to reproduce the structural varieties observed in ESI-IM-MS. The temperature effect on the gas-phase structures of folded proteins was theoretically and experimentally identified by using the variable temperature IM-MS;22,49 an increase in buffer-gas temperature up to 400 K causes a minor collapse of the folded structure, whereas a temperature increase up to 475 K induces protein unfolding. In this study, it was found that the tail regions of the histone multimers, rather than the core regions, collapsed significantly to varying degrees as the simulation temperature increased up to 400 K. Furthermore, it should be remembered that the ESI-IM-MS technique contains various subprocesses, the activities of which often have direct impacts on the temperature of the system. If MD simulation could be performed by taking other factors implicated in the complicated process of ESI-IM-MS into account, such as a gradual alteration

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CONCLUSION Gas-phase structures of the histone multimers, H2A/H2B dimer and (H3/H4)2 tetramer, with disordered tail regions were investigated by ESI-IM-MS and MD simulation. As a result, the structural properties of the histone multimers in the gas phase were interpreted as follows: (1) the overall sizes of the multimers were drastically reduced to two-thirds upon transition from solution into the gas phase; (2) the size reduction was mainly due to the compaction of the tail regions; (3) the extent of reduction observed for the tails was not uniform, which contributed to the variety of multimer CCS values observed in ESI-IM-MS; and (4) the range of distribution for experimentally observed CCS values was well covered by MD simulation across several temperatures. The present study showed a potential use for IM-MS in combination with the analysis of structure ensembles obtained by MD simulation to investigate the structures of protein assemblies with disordered regions. Although several issues are suggested to be solved, this combination is promising to characterize structures of IDPs that are difficult to crystallize. Furthermore, since it has been suggested that various modifications on the histone N-terminal tails induce dynamic structural changes of NCP leading to regulations of gene expression, structural characterization of the disordered tail regions in the modified NCP, such as methylated NCP, by the present method may help to reveal biological significance of the histone tail modification.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Materials and Methods, Supplementary Tables S-1 and S-2, Supplementary Figures S-1−S-6, and Supplementary Discussions, as mentioned in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Satoko Akashi or Yoshifumi Nishimura: e-mail, akashi@ tsurumi.yokohama-cu.ac.jp (S.A.); phone, +81-45-508-7217; fax, +81-45-508-7362. Present Addresses §

K.T.: Chugai Research Institute for Medical Science, Inc., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan. ∥ Y.A.: KISCO Ltd., 4-11-2 Nihonbashi Honcho, Chuo-ku, Tokyo 103-8410, Japan. Notes

The authors declare no competing financial interest. 4170

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(28) Saikusa, K.; Kuwabara, N.; Kokabu, Y.; Inoue, Y.; Sato, M.; Iwasaki, H.; Shimizu, T.; Ikeguchi, M.; Akashi, S. Analyst 2013, 138, 1441−1449. (29) Shimoyama, S.; Nagadoi, A.; Tachiwana, H.; Yamada, M.; Sato, M.; Kurumizaka, H.; Nishimura, Y.; Akashi, S. J. Mass Spectrom. 2010, 45, 900−908. (30) Tanaka, Y.; Tawaramoto-Sasanuma, M.; Kawaguchi, S.; Ohta, T.; Yoda, K.; Kurumizaka, H.; Yokoyama, S. Methods 2004, 33, 3−11. (31) Zhong, Y.; Hyung, S. J.; Ruotolo, B. T. Analyst 2011, 136, 3534−3541. (32) Giles, K.; Williams, J. P.; Campuzano, I. Rapid Commun. Mass Spectrom. 2011, 25, 1559−1566. (33) Downard, K. M.; Kokabu, Y.; Ikeguchi, M.; Akashi, S. FEBS J. 2011, 278, 4044−4054. (34) Ruotolo, B. T.; Benesch, J. L. P.; Sandercock, A. M.; Hyung, S. J.; Robinson, C. V. Nat. Protoc. 2008, 3, 1139−1152. (35) Tachiwana, H.; Kagawa, W.; Osakabe, A.; Kawaguchi, K.; Shiga, T.; Hayashi-Takanaka, Y.; Kimura, H.; Kurumizaka, H. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10454−10459. (36) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San Carlos, CA, 2002; http://www.pymol.org. (37) Ikeguchi, M. J. Comput. Chem. 2004, 25, 529−541. (38) Metropolis, N.; Rosenbluth, A W.; Rosenbluth, M. N.; Teller, A. H. J. Chem. Phys. 1953, 21, 1087−1092. (39) Miteva, M.; Demirev, P. A.; Karshikoff, A. D. J. Phys. Chem. B 1997, 101, 9645−9650. (40) Dobo, A.; Kaltashov, I. A. Anal. Chem. 2001, 73, 4763−4773. (41) Borysik, A. J. H.; Radford, S. E.; Ashcroft, A. E. J. Biol. Chem. 2004, 279, 27069−27077. (42) Testa, L.; Brocca, S.; Grandori, R. Anal. Chem. 2011, 83, 6459− 6463. (43) Benesch, J. L. P.; Ruotolo, B. T. Curr. Opin. Struct. Biol. 2011, 21, 641−649. (44) http://www.indiana.edu/∼nano/software.html. (45) Lee, S. W.; Freivogel, P.; Schindler, T.; Beauchamp, J. L. J. Am. Chem. Soc. 1998, 120, 11758−11765. (46) Steinberg, M. Z.; Breuker, K.; Elber, R.; Gerber, R. B. Phys. Chem. Chem. Phys. 2007, 9, 4690−4697. (47) Michaelevski, I.; Eisenstein, M.; Sharon, M. Anal. Chem. 2010, 82, 9484−9491. (48) Merenbloom, S. I.; Flick, T. G.; Williams, E. R. J. Am. Soc. Mass Spectrom. 2012, 23, 553−562. (49) Pacholarz, K. J.; Barran, P. E. 19th International Mass Spectrometry Conference, Kyoto, Japan, 2012; PMo-021.

ACKNOWLEDGMENTS This work was supported by the Grants-in-Aid for Scientific Research 22570120 (S.A.), the Grants-in-Aid for Scientific Research on Innovative Areas 21113003 (S.A.), and the Platform for Drug Discovery, Informatics, and Structural Life Science (H.K., M.I., and Y.N.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors are grateful to Kana Hara (Yokohama City University) for providing the H2A/H2B dimers for the IM-MS experiments. All computations were done on the Tsurumi campus of Yokohama City University, Yokohama, Japan.

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REFERENCES

(1) Luger, K.; Mäder, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J. Nature 1997, 389, 251−260. (2) Berger, S. L. Nature 2007, 447, 407−412. (3) Downs, J. A.; Nussenzweig, M. C.; Nussenzweig, A. Nature 2007, 447, 951−958. (4) Felsenfeld, G.; Groudine, M. Nature 2003, 421, 448−453. (5) Jenuwein, T.; Allis, C. D. Science 2001, 293, 1074−1080. (6) Strahl, B. D.; Allis, C. D. Nature 2000, 403, 41−45. (7) Luger, K. Chromosome Res. 2006, 14, 5−16. (8) Arents, G.; Burlingame, R. W.; Wang, B. C.; Love, W. E.; Moudrianakis, E. N. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10148− 10152. (9) Mittag, T.; Forman-Kay, J. D. Curr. Opin. Struct. Biol. 2007, 17, 3−14. (10) Bernadó, P.; Svergun, D. I. Mol. BioSyst. 2012, 8, 151−167. (11) Sugase, K.; Dyson, H. J.; Wright, P. E. Nature 2007, 447, 1021− 1025. (12) Kokabu, Y.; Murayama, Y.; Kuwabara, N.; Oroguchi, T.; Hashimoto, H.; Tsutsui, Y.; Nozaki, N.; Akashi, S.; Unzai, S.; Shimizu, T.; Iwasaki, H.; Sato, M.; Ikeguchi, M. J. Biol. Chem. 2011, 286, 43569−43576. (13) Miyagi, A.; Tsunaka, Y.; Uchihashi, T.; Mayanagi, K.; Hirose, S.; Morikawa, K.; Ando, T. ChemPhysChem 2008, 9, 1859−1866. (14) Uchihashi, T.; Kodera, N.; Ando, T. Nat. Protoc. 2012, 7, 1193− 1206. (15) Heck, A. J. R. Nat. Methods 2008, 5, 927−933. (16) Sharon, M.; Robinson, C. V. Annu. Rev. Biochem. 2007, 76, 167− 193. (17) Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1995, 117, 10141−10142. (18) Bohrer, B. C.; Merenbloom, S. I.; Koeniger, S. L.; Hilderbrand, A. E.; Clemmer, D. E. Annu. Rev. Anal. Chem. 2008, 1, 293−327. (19) Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Science 2005, 310, 1658−1661. (20) Uetrecht, C.; Rose, R. J.; van Duijin, E.; Lorenzen, K.; Heck, A. J. R. Chem. Soc. Rev. 2010, 39, 1633−1655. (21) Politis, A.; Park, A. Y.; Hyung, S. J.; Barsky, D.; Ruotolo, B. T.; Robinson, C. V. PLoS One 2010, 5, e12080. (22) Hall, Z.; Politis, A.; Bush, M. F.; Smith, L. J.; Robison, C. V. J. Am. Chem. Soc. 2012, 134, 3429−3438. (23) Mao, Y.; Ratner, M. A.; Jarrold, M. F. J. Phys. Chem. B 1999, 103, 10017−10021. (24) Bernstein, S. L.; Wyttenbach, T.; Baumketner, A.; Shea, J. E.; Bitan, G.; Teplow, D. B.; Bowers, M. T. J. Am. Chem. Soc. 2005, 127, 2075−2084. (25) Baumketner, A.; Bernstain, S. L.; Wyttenbach, T.; Bitan, G.; Templow, D. B.; Bowers, M. T.; Shea, J. E. Protein Sci. 2006, 15, 420− 428. (26) Bernstein, S. L.; Dupuis, N. F.; Lazo, N. D.; Wyttenbach, T.; Condron, M. M.; Bitan, G.; Teplow, D. B.; Shea, J. E.; Ruotolo, B. T.; Robinson, C. V.; Bowers, M. T. Nat. Chem. 2009, 1, 326−331. (27) Pagel, K.; Natan, E.; Hall, Z.; Fersht, A. R.; Robinson, C. V. Angew. Chem., Int. Ed. 2013, 52, 361−365. 4171

dx.doi.org/10.1021/ac400395j | Anal. Chem. 2013, 85, 4165−4171