Structural Diversity of Nucleosomes Characterized by Native Mass

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Structural Diversity of Nucleosomes Characterized by Native Mass Spectrometry Kazumi Saikusa, Akihisa Osakabe, Daiki Kato, Sotaro Fuchigami, Aritaka Nagadoi, Yoshifumi Nishimura, Hitoshi Kurumizaka, and Satoko Akashi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01649 • Publication Date (Web): 02 Jun 2018 Downloaded from http://pubs.acs.org on June 2, 2018

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Analytical Chemistry

Structural Diversity of Nucleosomes Characterized by Native Mass Spectrometry

Kazumi Saikusa1, 2, Akihisa Osakabe3†, Daiki Kato3, Sotaro Fuchigami1¶ , Aritaka Nagadoi1, Yoshifumi Nishimura1, Hitoshi Kurumizaka3§ , and Satoko Akashi1*

1

Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

2

Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan

3

Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan †

Present address: Gregor Mendel Institute, Frederic Berger Lab., Dr. Bohr-Gasse 3, 1030 Vienna, Austria

¶

Present address: Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo, Kyoto, 606-8502, Japan

§

Present address: IQB, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan

* Correspondence to: Satoko Akashi [email protected] Tel: +81-45-508-7217, FAX: +81-45-508-7362 Running title: Structural diversity of nucleosomes

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 classed 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, 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 collision cross-section (CCS) values have slightly loosened structure with extended tail regions, which might relate to the biological function of histone tails.

Keywords: histone tail; intrinsically disordered region; electrostatic effect; charge states; electrospray ionization ion mobility mass spectrometry.


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Introduction Eukaryote genetic information is compactly stored in nuclei, and the basic structural unit of DNA packing is the nucleosome core particle (NCP). The NCP has one histone octamer consisting of two molecules each of H2A, H2B, H3, and H4, as well as ca. 147 base pairs (bp) of DNA. All four histone proteins have functional intrinsically disordered regions (IDRs), i.e., histone tails. These histone N-tail regions, which consist of ca. 20–45 amino acids, are rich in basic amino acids and protrude from the NCP core structure [1]. They enable target recognition and signal transduction in concert with various N-tail modifications [2–8]. Although histone tails play important roles in the regulation of DNA transcription, replication, and repair [2–8], they are not clearly observable in the X-ray structure because of their flexibility [1]. As is the case for histone tails, various IDRs of proteins, especially in eukaryotes, are involved in critical biological functions [9–13]. When IDRs encounter a target, they change conformation and form complexes with the target; thus, the lack of a definite structure is an advantage for these proteins. Because of the flexibility of protein structures having IDRs, analyzing them by X-ray crystallography is impractical. If they are relatively small (35+, and >36+ charged ions for mononucleosomes with 250, 294, and 342 bp DNA, respectively) cannot be clearly observed in Figure 3. This result suggests that the flexible region of DNA is responsible for disappearance of highly charged ions. This is discussed further in following sections. We also attempted IM-MS of the mononucleosomes, but the ion intensity was not sufficient to accurately analyze the CCS of each ion (data not shown).

Dinucleosomes We then carried out native MS of reconstituted dinucleosomes with 294 and 342 bp DNA. Figure 4 shows the ESI mass spectra of the dinucleosomes in 50 mM ammonium acetate 11 ACS Paragon Plus Environment


solution. Since these DNA fragments contain two Widom 601 sequences, they can theoretically retain up to two histone octamers. The observed mass for the dinucleosome with 294 bp was 407,560±54, as previously reported [22], while that for the dinucleosome with 342 bp was 439,711±31. These mass values are consistent with the calculated masses for the two dinucleosomes, 405,516 (294 bp DNA) and 435,237 (342 bp DNA), respectively. The dinucleosome with 294 bp DNA gave intense signals corresponding to charges of 35+ to 45+ at m/z 9000–12000 and weak signals for charges of ≥46+ at m/z 7000–9000. In contrast, the dinucleosome with 342 bp DNA, which has a 48 bp linker region between two Widom 601 sequences (Figure S-2), presented ions with charges of 35+ to 45+, but ions with charges of ≥46+ were not apparent. The ESI mass spectrum of the dinucleosome with 342 bp suggests that the tails and free DNA linkers were in a compact form, resulting in the absence of ions at higher charge states. In contrast, if dinucleosomes have no flexible DNA linker, as is the case with canonical NCPs and OLDNs, higher charged ions were observed at m/z 7000–9000 for the dinucleosome with 294 bp, in addition to the major ions at lower charge states. We also attempted IM-MS of the two dinucleosomes, but the ion intensity was not sufficient to analyze the CCS of each ion in the IM-MS mode (data not shown).

Computational Structure Simulation of canonical NCP To evaluate the ESI-MS and ESI-IM-MS NCP results, MD simulations were performed for the canonical NCP in 50 or 500 mM NaCl solutions, and then two types of structural calculation were carried out in vacuo (as shown in Supplementary Scheme S-1). From the MD simulations of NCP in solutions, 10 structures for each NaCl concentration were obtained with CCSs of 10,210–10,460 Å2 (50 mM NaCl) and 11,310–11,640 Å2 (500 mM NaCl) (Figure 5 and Supplementary Figures S-7 and S-8). We found that the NCP core in the structural models 12 ACS Paragon Plus Environment

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obtained by MD simulation in solution was slightly loosened compared with the NCP core in the X-ray structure. The MD-simulated structures were subsequently subjected to MD simulation in vacuo, with 28+, 35+, or 45+ charges attached, which yielded 100 structures for each charge state for a total of 300 structures. The calculated CCS values for 300 structures were 7,350–7,900 Å2 (50 mM NaCl) and 7,470–8,290 Å2 (500 mM NaCl). These values were small compared with the calculated CCS value of 8,580 Å2 for the NCP X-ray structure (PDB: 3AFA), which presented a part of two histone H3 tails. By detailed examination of the theoretical structures obtained by MD simulation in vacuo, we found that histone tails were attached to the core region and the DNA structure wrapped around the core collapsed considerably, resulting in insufficient space for the histone octamer (Figure S-7). It should also be noted that there was no big difference in size even if the charge states were changed (Figure S-8). Next, we examined the other structural calculation in vacuo; the dehydrated structures were generated by 106-step energy minimization [36, 37]. Ten structures obtained by solution MD simulation in 50 or 500 mM NaCl solution were used to generate dehydrated structures with energy minimization, with 28+, 35+, or 45+ charges attached. The structures of the histone octamer in the core of dehydrated NCP likely changed little from the solution structural models, but the DNA strand apparently was positioned slightly closer to the core than that in the solution structure. The distance between the two DNA loops in the core region was shortened; the thickness of the core region apparently decreased (Figure S-7). The calculated CCSs of the dehydrated structures were 8,760–9,250 Å2 (50 mM NaCl) and 9,940–10,460 Å2 (500 mM NaCl) (Figures S-7 and S-8). The experimentally obtained CCS values of the 28+ charged ion was 8,630 Å2, which was ~400 Å2 smaller than that of the dehydrated structure produced from the 50 mM NaCl solution structure; it was almost equal to that of the X-ray structure of NCP (PDB: 3AFA), in which most of the histone tail regions are invisible. Meanwhile, experimental CCS for the 35+ 13 ACS Paragon Plus Environment


charged ion was 9,500 Å2, which was positioned between two dehydrated structures originating from the 50 and 500 mM NaCl solution structures. The CCS of the 45+ charged ion was larger than that of the MD-simulated structures in solution in the presence of 50 mM NaCl but smaller than that in 500 mM NaCl solution. It is likely that the largest conformers, C, with >41+ charge observed in IM-MS would also have extended or loosened tail regions attached to the loosened core structure, as demonstrated by MD simulation of NCP in solution (Figure S-7).

Discussion Using native MS, we characterized nucleosomes, large protein–DNA complexes reconstituted with histone proteins and DNA with various lengths. By detailed analysis of the results, we found that most nucleosomes have rather compact structures in the gas phase, but the existence of free DNA regions affected the range of charge state distribution. NCP and OLDN, in which the entire DNA regions were involved in the core formation, showed wide charge distributions, while mono- and dinucleosomes with free DNA regions showed a narrow range of charge distributions. Theoretical structural calculations suggest that the wide charge distributions were due to the structural diversity of the histone tail regions in addition to the core part. NCP has a folded core and flexible tail regions, as well as a structure that is stabilized mainly by electrostatic interactions between DNA and basic histone proteins. Therefore, a low ionic strength is advantageous for stabilizing protein–DNA complexes but not for stabilizing the histone octamer, which is located at the center of the NCP [1] and is stabilized mainly by hydrophobic interactions. Canonical NCPs gave intense signals of low-charge ions at m/z 6800– 9000. These ions correspond to the compact NCP structure with unextended tail regions, as well as weak signals of high-charge ions at m/z 4000–6800, which suggest a variety of conformations (Figure 1a), as observed in the theoretical structural calculation. Since we have applied moderate 14 ACS Paragon Plus Environment

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parameters for ESI, these highly charged ions were not products of the harsh experimental conditions. Similar findings were also obtained for OLDN, which consists of 250 bp DNA, a histone octamer, and a histone hexamer (Figure 2a). These results were indicated also by ESI-IM-MS (Figures 1b and 2b, Supplementary Figures S-3 and S-6). Observation of higher-charge-state ions for the reconstituted NCPs indicates that a small population of NCPs are large in size. This is true also for NCPs at a high ionic strength, such as those in 1 M ammonium acetate solution. That is, a high concentration of ammonium acetate basically did not affect the overall structure of the NCPs. ESI-MS of the mono- and dinucleosomes suggested “extra” DNA regions uninvolved in the direct interaction with the basic histone proteins diminished the ions at high charge states in the mass spectra (Figure 3). This result indicates that both the histone tails and extra DNA regions within mononucleosomes are not elongated but are in a compact form in the gas phase. In contrast, observed charge states of dinucleosomes varied according to the length of the DNA fragment (Figure 4). Within the dinucleosome reconstituted with 294 bp DNA, the entire DNA region may be involved in direct interaction with two histone octamers, as it was with the canonical NCP. In the ESI mass spectrum, we observed ions at high charge states with low intensity, in addition to the major peaks at low charge states. In contrast, in the case of dinucleosome with 342 bp DNA, it would be in a compact form, leading to the absence of high-charge ions in the ESI mass spectra. Considering the ESI-MS results for NCPs, OLDNs, and mono- and dinucleosomes, a linker DNA length of 48 bp might be long enough to reduce the structural diversity of nucleosomes in the gas phase. Figure 6 summarizes the relationship between molecular masses and observed ranges of charge states of nucleosomes and those of globular proteins based on ESI-MS [14]. The plot for the range of charge states versus mass for standard globular proteins has a logarithmic trend line, 15 ACS Paragon Plus Environment


with the range of charge states gradually increasing with the increment of molecular mass for large proteins. The trend of mononucleosomes with long DNA strands and dinucleosomes with a linker DNA is similar to that of globular proteins. In contrast, canonical NCPs (147 bp), OLDNs (250 bp), and dinucleosomes with 294 bp DNA had a range of charge states wider than that of globular proteins with the same molecular mass. To provide a comprehensive description of all data, the results are interpreted as follows: 1) Most histone tails and free DNA regions in NCPs and mono, and dinucleosomes have a compact structure in the gas phase. 2) A small population of the canonical NCPs and OLDNs can carry additional positive charges, leading to a variety of structures. 3) The extra DNA region that is not directly involved in NCP formation may be able to interact with itself, the histone tails, and the core regions in the gas phase, thereby resulting in compact structures. Tails of NCPs in solution may take a range of conformations having various degrees of contact with the core, but the population of NCPs with extended tails would be small. This can be supported not only by the MD simulation results in the present study, but also by previous simulation results in explicit water [45, 46]. To investigate the structures corresponding to the ions in ESI-MS of NCP, MD simulation in solution and gas phase was carried out in the present study. However, a single set of simulations cannot represent the structure observed in the ESI mass spectra. This was likely due to the greater difficulty in reproducing the positive-mode ESI process of protein–DNA complexes by computational simulation, as compared with the case of protein–protein complexes. For ESI-MS, samples in aqueous solutions at neutral pH were prepared. Thus, NCP molecules were solvated, and dissociated ionic groups were paired with counter ions existing in the solution. In MD simulations for NCP in solution, these charged states were reproduced, and calculations that included NaCl were performed. Upon ESI, desolvation occurs concomitantly 16 ACS Paragon Plus Environment

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with ionization. To replicate the ESI process in the positive ion mode, we then carried out gas-phase structural calculations; 28, 35, or 45 positive charges were attached to the NCP solution structural models, and they were used for in vacuo calculations. That is, removal of solvents and change in the charge states were achieved simultaneously and instantaneously, and then gas-phase calculations were carried out. For globular protein complexes, MD simulation programs and force fields have been explored and optimized by many studies [20, 36, 37, 41, 47, 48]. However, it is presently difficult to reproduce experimental results by calculation with a simple program; this was demonstrated by Gabelica et al. for short DNA oligomers (>36 bp), which were easily compacted in the gas phase [49]. The experimental CCS values for the NCP major conformer A were similar to the calculated CCS of the X-ray structure of PDB:3AFA, in which histone tail regions were almost invisible. Meanwhile, the dehydrated structure of NCP originating from a 50 mM NaCl solution structure showed a slightly larger CCS value than the experimental measurement. Thus, the structure of conformer A would be similar to this dehydrated structure, with histone tails more firmly wrapped around the core region. Besides, considering that MD simulations in the gas phase suggest considerable structure distortion (Figure S-7), a slightly distorted dehydrated structural model originating from the 50 mM NaCl solution would correspond to the major conformer A. Meanwhile, the conformer C, which had the largest CCS values, would probably be represented by an MD-simulated structure in solution, in which the histone tail regions are extended on the slightly loosened core. In MD-simulated structural models in the 50 mM NaCl solution, N-tail regions of H3 and H2B are especially extended among four histone proteins; N-tail of H2A would also be slightly extended. Considering that modifications of N-tail regions of H3 and H2B play particularly significant roles in controlling transcription initiation, it would be reasonable to assume that these N-tail regions are extended outwards in the calculated 17 ACS Paragon Plus Environment


structural models, leading to epigenetic modification. The middle-sized conformer B would have similar core structure with the dehydrated structural models generated from 50 mM or 500 mM NaCl solution. When the theoretical structures originated from 50 mM and 500 mM NaCl solution are compared, it seems that there is little difference in the core region structures but extension levels of histone tails are different (Figures 5 and S-7). That is, histone tail regions are widely spread in the structures originating from 500 mM NaCl solutions (Figures S-7 (b) and (d)) but they are positioned close to the core in the structures originating from 50 mM NaCl (Figures S-7 (a) and (c)). The widths of CCS distributions of conformers B and C might correspond to the structural diversity of histone tail regions; the narrow distribution width for conformer A would imply that the histone tails are not extended but in a compact form. NCP is a very complex system; a globular protein core is surrounded by DNA, and intrinsically disordered tails are extended from the core region. In solution, phosphate groups of DNA and basic and acidic amino acids are dissociated, and free DNA and histone tail regions are disordered. Since excess ammonium and acetate ions exist in the sample solution during the electrospray ionization process, they would be the main counter ions for phosphate and side chains of amino acids. Upon ionization in the positive ion mode, desolvation occurs with gradual removal of volatile molecules, as well as shrinkage of histone tails and free DNA. In the case of canonical NCP and OLDN, most of the histone tail regions shrunk along with slight distortion of the core region at ionization, but a small population of histone tails remained elongated while retaining many positive charges. If flexible free DNA regions were present in nucleosomes, their negative charges were diminished by ionization in the positive ion mode. Under these conditions, it would be possible for the free DNA region to interact with itself and the core region, while also catching the histone tails in the middle. Consequently, ions with high charge for nucleosomes having a long histone-free DNA region were not observed in the low-m/z region. To replicate 18 ACS Paragon Plus Environment

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these processes, we performed computational calculations for the mononucleosome, but they were unsuccessful in giving feasible structural models representing a smooth transition from solution to the gas phase. As mentioned, our results suggest that the diversity of nucleosome structures in the gas phase may be affected in the presence of histone-free DNA regions. When eukaryotic chromatin is unfolded and viewed under an electron microscope, it gives a beads-on-a-string appearance; that is, NCPs are linked via histone-free DNA strands. Transcription initiation is controlled by various modifications of histone tail regions (acetylation, methylation, phosphorylation, etc) [2– 8]. With these modifications, altering the electrostatic states of histone tails is possible. Modified histone tails within nucleosomes with long DNA strands might behave differently from nucleosomes with unmodified histone tails. For example, acetylation reduces the basicity of histone tails, and acetylated tails may have less contact with histone-free DNA and are probably be more flexible than unmodified histone tails in nucleosomes. Because of their enhanced flexibility, acetylated histone tails can be easily recognized by chromatin remodeling factors. Although further study is required, our findings that histone-free DNA regions affect the structural diversity of nucleosomes may provide biological insight into the first step of regulation of eukaryotic transcription initiation.

Acknowledgments This work was partly supported by the Platform Project for Supporting in Drug Discovery and Life Science Research from the Japan Agency for Medical Research and Development (AMED) grant numbers JP17am0101076 (to HK and SA), JP17am0101073 (to



YN), and JP17am0101109 (to SF) and JSPS KAKENHI grant numbers JP26505009 (to SA), JP17K07313 (to SA), JP16K18528 (to KS), JP26104531 (to SF), and JP25116002 (to HK).

Supporting Information Supplementary Methods, Scheme S-1, Table S-1, and Figures S-1−S-8 are available at http://pubs.acs.org.

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40. Kaltashov, I.A.; Mohimen, A. Anal Chem. 2005, 77, 5370-5379. 41. Pagel, K.; Natan, E.; Hall, Z.; Fersht, A.R.; Robinson, C.V. Angew Chem Int Ed Engl. 2013, 52, 361-365. 42. Laszlo, K.J.; Munger, E.B.; Bush, M.F. J Am Chem Soc. 2016, 138, 9581-9588. 43. Jhingree, J.R.; Bellina, B.; Pacholarz, K.J.; Barran, P.E. J Am Soc Mass Spectrom. 2017, 28, 1450-1461. 44. Beveridge R.; Chappuis, Q.; Macphee, C.; Barran, P. Analyst 2013, 138, 32-42. 45. Shaytan, A.K.; Armeev, G.A.; Goncearenco, A.; Zhurkin, V.B.; Landsman, D.; Panchenko, A.R. J Mol Biol. 2016, 428, 221-237. 46. Erler, J.; Zhang, R.; Petridis, L.; Cheng, X.; Smith, J.C.; Langowski, J. Biophys J. 2014, 107, 2911-2922. 47. Landreh, M.; Marklund, E.G.; Uzdavinys, P.; Degiacomi, M.T.; Coincon, M.; Gault, J.; Gupta, K.; Liko, I.; Benesch, J.L.; Drew, D.; Robinson, C.V. Nat Commun. 2017, 8, 13993. 48. Devine, P.W.A.; Fisher, H.C.; Calabrese, A.N.; Whelan, F.; Higazi, D.R.; Potts, J.R.; Lowe, D.C.; Radford, S.E.; Ashcroft, A.E. J Am Soc Mass Spectrom. 2017, 28, 1855-1862. 49. Porrini, M.; Rosu, F.; Rabin, C.; Darré, L.; Gómez, H.; Orozco, M.; Gabelica, V. ACS Cent Sci. 2017, 3, 454-461.



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Figure Legends Figure 1. ESI mass spectrum (a) and IM-MS 2D contour plot (b) of the canonical NCP. A sample in 50 mM ammonium acetate solution was prepared. Structures determined by X-ray crystallography (PDB: 3AFA) are also indicated from two directions (c). Yellow, red, blue, and green strands correspond to histones H2A, H2B, H3, and H4, respectively, and white line models represent DNA. Structures were generated using PyMol software [39].

Figure 2. ESI mass spectrum (a) and IM-MS 2D contour plot (b) of OLDN. A sample in 50 mM ammonium acetate solution was prepared.

Structures determined by X-ray

crystallography (PDB: 5GSE) are also indicated from two directions (c). Yellow, red, blue, and green strands correspond to histones H2A, H2B, H3, and H4, respectively, and white line models represent 250 bp DNA. Structures were generated using PyMol software [39].

Figure 3. ESI mass spectra of mononucleosomes composed of histone proteins and 250 bp (a), 294 bp (b), or 342 bp (c) of DNA. Samples in 50 mM ammonium acetate solution were prepared. Gray arrowheads and solid arrows indicate multiply charged ions of mononucleosomes and double-stranded DNA, respectively.

Figure 4. ESI mass spectra of dinucleosomes composed of histone proteins and 294 bp (a) or 342 bp (b) of DNA. Samples in 50 mM ammonium acetate solution were prepared. Gray arrowheads indicate multiply charged ions of dinucleosomes.

Figure 5. Superimposed plots of CCS values obtained by ESI-IM-MS and structural calculation


Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of the canonical NCP. Experimental CCS distributions were generated using the Origin v.8.6 program (OriginLab Corporation, Northampton, MA) and based the original data in Figure S-3. CCS values of the structural models obtained by MD simulation in 50 mM and 500 mM NaCl solutions and those obtained by dehydration and energy minimization (initial structures: originated from solution structures in 50 mM and 500 mM NaCl) are summarized in Figure 5 superimposed on the experimentally obtained CCS distributions. Representative structural models obtained by energy minimization of dehydrated structures originating from the MD-simulated structures in 50 mM and 500 mM NaCl solution, and those obtained by MD simulations in the presence of 500 mM NaCl solution are indicated below and are accompanied with X-ray structures of NCP of PDB:3AFA and PDB:1KX5.

Figure 6. Plots of charge state range versus calculated molecular mass for nucleosomes and globular protein standards. The data point for canonical NCP, indicated by a blue square, is derived from the ESI mass spectrum of the NCP in Figure 1. The data point for OLDN, indicated by a light blue diamond, is derived from the ESI mass spectrum in Figure 2. Data for mono- and dinucleosomes, indicated by yellow triangles and red circles, are derived from the ESI mass spectra in Figures 3 and 4. Data for globular protein standards, indicated by blue crosses, originated from the study of Beveridge et al. [14]. The solid line corresponds to the trend line for globular protein standards.



Figure 1. ESI mass spectrum (a) and IM-MS 2D contour plot (b) of the canonical NCP. A sample in 50 mM ammonium acetate solution was prepared. Structures determined by X-ray crystallography (PDB: 3AFA) are also indicated from two directions (c). Yellow, red, blue, and green strands correspond to histones H2A, H2B, H3, and H4, respectively, and white line models represent DNA. Structures were generated using PyMol software [39]. 232x233mm (72 x 72 DPI)

ACS Paragon Plus Environment

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Figure 2. ESI mass spectrum (a) and IM-MS 2D contour plot (b) of OLDN. A sample in 50 mM ammonium acetate solution was prepared. Structures determined by X-ray crystallography (PDB: 5GSE) are also indicated from two directions (c). Yellow, red, blue, and green strands correspond to histones H2A, H2B, H3, and H4, respectively, and white line models represent 250 bp DNA. Structures were generated using PyMol software [39]. 231x233mm (72 x 72 DPI)



Figure 3. ESI mass spectra of mononucleosomes composed of histone proteins and 250 bp (a), 294 bp (b), or 342 bp (c) of DNA. Samples in 50 mM ammonium acetate solution were prepared. Gray arrowheads and solid arrows indicate multiply charged ions of mononucleosomes and double-stranded DNA, respectively. 231x170mm (72 x 72 DPI)


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Figure 4. ESI mass spectra of dinucleosomes composed of histone proteins and 294 bp (a) or 342 bp (b) of DNA. Samples in 50 mM ammonium acetate solution were prepared. Gray arrowheads indicate multiply charged ions of dinucleosomes 234x122mm (72 x 72 DPI)



Figure 5. Superimposed plots of CCS values obtained by ESI-IM-MS and structural calculation of the canonical NCP. Experimental CCS distributions were generated using the Origin v.8.6 program (OriginLab Corporation, Northampton, MA) and based the original data in Figure S-3. CCS values of the structural models obtained by MD simulation in 50 mM and 500 mM NaCl solutions and those obtained by dehydration and energy minimization (initial structures: originated from solution structures in 50 mM and 500 mM NaCl) are summarized in Figure 5 superimposed on the experimentally obtained CCS distributions. Representative structural models obtained by energy minimization of dehydrated structures originating from the MDsimulated structures in 50 mM and 500 mM NaCl solution, and those obtained by MD simulations in the presence of 500 mM NaCl solution are indicated below and are accompanied with X-ray structures of NCP of PDB:3AFA and PDB:1KX5. 169x116mm (300 x 300 DPI)


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Figure 6. Plots of charge state range versus calculated molecular mass for nucleosomes and globular protein standards. The data point for canonical NCP, indicated by a blue square, is derived from the ESI mass spectrum of the NCP in Figure 1. The data point for OLDN, indicated by a light blue diamond, is derived from the ESI mass spectrum in Figure 2. Data for mono- and dinucleosomes, indicated by yellow triangles and red circles, are derived from the ESI mass spectra in Figures 3 and 4. Data for globular protein standards, indicated by blue crosses, originated from the study of Beveridge et al. [14]. The solid line corresponds to the trend line for globular protein standards. 251x162mm (72 x 72 DPI)


Structural Diversity of Nucleosomes Characterized by Native Mass

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