Probing the Binding Interfaces of Histone-Aptamer by Photo Cross

Dec 8, 2016 - We successfully achieved specific enrichment of histone H4 and further developed a new analysis strategy for histone-aptamer interaction...
62 downloads 21 Views 1MB Size
Download PDF
Letters pubs.acs.org/acschemicalbiology

Probing the Binding Interfaces of Histone-Aptamer by Photo CrossLinking Mass Spectrometry Congcong Lu,† Shanshan Tian,‡ Guijin Zhai,‡ Zuofei Yuan,§ Yijun Li,† Xiwen He,† Yukui Zhang,†,∥ and Kai Zhang*,‡,† †

Department of Chemistry, Nankai University, Tianjin 300071, People’s Republic of China Tianjin Key Laboratory of Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, People’s Republic of China § Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States ∥ Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Histone proteins, which could interact with DNA, play important roles in the regulation of chromatin structures, transcription, and other DNA-based biological processes. Here, we developed a novel aptamer-based probe for the analysis of histone H4-aptamer interfaces. This probe contains a DNA sequence for specific recognition of histone H4, a biotin tag for affinity enrichment, an aryl azide photoactive group for cross-linking and a cleavable disulfide group to dissociate aptamer from labeled histones. We successfully achieved specific enrichment of histone H4 and further developed a new analysis strategy for histoneaptamer interaction by photo cross-linking mass spectrometry. The binding area of histone H4 to aptamer was investigated and discussed for the first time. This strategy exhibits great potential and might further contribute to the understanding of histone−DNA interaction patterns.

I

It has been proved that protein−DNA interactions are involved in different biological processes. The analysis of protein−DNA binding is an important aspect in understanding those processes on a molecular biology level, such as replication, transcription, and genetic recombination.1,8 Histones, as a notable example, interact with DNA to help the formation of chromatin assembly.8 There are many biochemical, biophysical, and bioinformatics techniques to determine protein−DNA interactions (such as chromatin immunoprecipitation and H/D exchange mass spectrometry (H/DXMS)).9−11 However, these methods usually require rather stringent experimental conditions. Take H/DX-MS for example, this strategy requires highly pure proteins and the rate of hydrogen exchange between a protein and bulk solvent could be influenced by many environmental factors. Considering that the N-terminal tail of H4 protrudes outside of the histone core, the exchange reaction happens very fast and easily at histone N-terminal tails,12 and it might be still difficult to block H/D exchange even after binding to other molecules. Different from the above strategies, aptamer assay is a recently well-developed technique, requiring less complicated operation

n eukaryotic cells, the nucleosome consists of 147 base pairs of DNA wrapped around an octamer of core histones (H2A, H2B, H3, and H4).1,2 Histones, as the main complement of chromatin, can affect chromatin structures and therefore influence diverse biological processes by interacting with DNA or proteins through various post-translational modifications (PTMs).1,3 The identification of histones and their PTMs has become a high priority in current epigenetic studies. To explore those questions in greater detail, it is needed to get a unitary histone. At present, a single histone is typically purified by HPLC-based or electrophoresis-based isolation from acidextraction or salt-extraction mixtures, which is complicated and time-consuming.4 Therefore, a fast and effective method to get one single histone is highly desirable. Compared to antibodies, aptamers are more stable, much cheaper, and easier to obtain. Thus, aptamers rival antibodies in many analytical applications and are widely used in protein recognition and detections.5,6 In 2009, Chaput et al. reported a novel aptamer with high affinity to K16 acetylated histone H4,7 and then Shao et al. developed an aptamer-based approach to enrich histone proteins.5 This method can extract histone mixtures from whole cell lysate. However, it is still difficult to isolate H4 from histones. Moreover, many basic questions need to be answered about how the aptamer interacts with histone H4. © 2016 American Chemical Society

Received: September 14, 2016 Accepted: December 8, 2016 Published: December 8, 2016 57

DOI: 10.1021/acschembio.6b00797 ACS Chem. Biol. 2017, 12, 57−62

Letters

ACS Chemical Biology

Scheme 1. Analysis Strategy for Probing Histone H4-Aptamer Binding Interfaces: (I) Aptamer-Based Selective Separation and Enrichment of Histone H4 and (II) Aptamer-Based Photo Cross-Linking of Histone H4

biotin label transfer reagent, structure can be found in Figure S1A) by NH2/NHS reaction, after which three functional groups were added at the 5′-end (or 3′-end) of aptamer, respectively. Then, modified aptamer could selectively interact with histone H4 when incubated with histone mixtures. After affinity enrichment, target proteins were eluted and determined by SDS-PAGE as shown in path I. While in alternative path II, photo cross-linking reaction was initiated by UV light, and then DTT was used to open the disulfide bond. Thus, only photo cross-linked proteins were enriched. Then, targeted protein histone H4 was further analyzed by high resolution MS. By comparing the identification information before and after photo cross-linking, the binding area of histone H4 to aptamer could be characterized. In this study, we used histone mixtures obtained by acid extraction as samples to investigate the binding area of histone H4 and aptamer. First, histones were separated by 15% SDSPAGE and stained by coomassie blue to check the extraction efficiency. As shown in Figure 1A, five kinds of histone isoforms were all extracted with a high extraction efficiency, including histone H4. Then, we investigated the selectivity and binding affinity of this aptamer probe by path I. Owing to the highly selective and stable interaction (Kd ∼ 10−14 M) under a wide range of experimental, streptavidin−biotin systems is one of the most widely used techniques in affinity enrichment.17 Here, we first examined the enrichment efficiency of histone H4 using streptavidin agarose beads. Proteins captured without and with a 5′-end modified aptamer were separated by SDS-PAGE and then silver-stained. As we can see in Figure 1B, there was a significant band in lane B1 (without aptamer for negative control) just at the same location of histone H4 (lane B2). To

procedures due to its great specificity and affinity. By changing oligonucleotide sequence, the ligand binding sites of a DNA aptamer that are complementary in shape and charge to a desired protein can be determined. The binding sites of proteins are rarely studied. Therefore, development of a sensitive and reliable method for studying protein−aptamer interactions, especially binding sites of proteins, would be of great significance. During the past decade, cross-linking combined with mass spectrometry (XL-MS) studies have been successfully undertaken to investigate purified proteins and protein complexes, providing information on both the structure and interactions of proteins.13−15 However, few attempts have focused on the investigation of protein−DNA interaction. Here, we designed a novel multiple functional chemical probe, which contains a new aptamer with high specific recognition capability for histone H416 (sequence information can be found in the Supporting Information) and further developed a novel strategy for analysis of histone−aptamer interaction by photo XL-MS. In this study, we successfully achieved a specific separation of histone H4 and characterized the binding area of H4 to aptamer at N-terminal tails. According to our best knowledge, this is the first report about probing the binding interfaces of histone H4-aptamer using aptamer-based photo XL-MS. This method is highly attractive for the study of protein−DNA interactions. In fact, the analysis of histone−DNA interactions will contribute to the understanding of histone mechanism in the regulation of chromatin structures and other important cell processes. The principle of this approach is briefly shown in Scheme 1 (details can be found in the Supporting Information). The aptamer was first modified with SBED reagent (sulfo-SBED 58

DOI: 10.1021/acschembio.6b00797 ACS Chem. Biol. 2017, 12, 57−62

Letters

ACS Chemical Biology

From former results through path I in Scheme 1, it can be easily found that the aptamer used here showed great selectivity and binding capacity toward histone H4, which was the base of the following study of histone H4−aptamer interaction. As shown in Scheme 1, three functional groups were linked to the aptamer after chemical modification, including a photoactive cross-linker aryl azide. When this cross-linker is exposed to lone wave UV light (330−370 nm), it forms a nitrene group that can initiate addition reactions with double bonds, insertion into C− H and N−H sites without specificity, or subsequent ring expansion to react as a nucleophile with primary amines.22 Those covalent modifications in protein structure will lead to a mass shift in both MS and MS/MS spectra and further result in an unrecognized peptide sequence (shown in black in path II) because the insertion is random and hard to detect in MS/MS. By comparing the identification information before and after photo cross-linking, we can reasonably infer the binding area of histone H4 to aptamer, which is the first time the binding area of histone proteins by the use of aptamer-based photo XL-MS has been studied. To probe the binding interfaces of histone−aptamer, we first determined the efficiency of photo cross-linking of histone H4 (shown in Figure 2). In lane 3, UV light was added to initiate

Figure 1. SDS-PAGE results of (A) histones mixture, coomassie blue staining; (B) proteins captured by streptavidin agarose beads (1) without aptamer and (2) with 5′-end modified aptamer, silver staining; (C) proteins captured by monoavidin agarose beads (1) without aptamer and (2) with 5′-end modified aptamer, silver staining. All gels were run under the same condition. B and C shared the same MW markers.

characterize the contamination, the band in lane B1 was excised and then digested by trypsin. Further MS/MS identification indicated that protein in lane B1 was streptavidin denatured by the harsh elution conditions (Figure S2). Results of SDS-PAGE and MS/MS both showed that a streptavidin−biotin system was not an appropriate option due to a similar molecular weight of denatured avidin with histone H4. Compared to the strong streptavidin−biotin interaction, monoavidin has a weaker interaction with biotin (Kd ∼ 10−7 M), which can reversibly bind to biotin and release targets under a mild elution condition.18 We next examined the enrichment efficiency of histone H4 using a monoavidin−biotin system, and results are shown in Figure 1C. Apparently, there was no protein captured when without aptamer (lane C1). MS/ MS identification indicated that protein in lane C2 was histone H4 (Figure S3), and no avidin was eluted. When there is a larger amount of salt, the separation efficiency of SDS-PAGE will be affected, leading to a diffuse protein band. Therefore, to get a relative better result, we optimized the elution method (Figure S4). Combining three times eluting solution eluted by biotin buffer was chosen as the most appropriate elution method in all experiments unless otherwise mentioned. More details can be found in supplemental experiments in the Supporting Information. Besides the internal influence of base arrangement, the structure of oligonucleotides is closely linked with outside factors, such as temperature, pH, and salt concentration.19,20 All those factors would have an effect on the efficiency of affinity enrichment, as well as the length of the linker.21 The predicted secondary structure showed that the aptamer used here would adopt a third stem-loop motif when increasing the salt concentration (Figure S1C and D), causing a different recognition capability and binding interaction to histone H4,16 as well as changes in arm length of the linker. Thus, we investigated the influence of salt concentration and length of linker on the efficiency of selective separation and enrichment of histone H4 by the use of 5′-end and 3′-end modified aptamers in three different buffers (low, middle, and high salt binding buffer). As shown in Figure S5, both kinds of modified aptamer can successfully recognize and bind to histone H4 no matter which kind of salt concentration was used. SDS-PAGE results indicated that this modified aptamer can be widely used in different conditions with a rather good recognition and strong binding captivity.

Figure 2. SDS-PAGE results of aptamer-based photo cross-linking of histone H4, silver staining. UV: + light added, − without light. DTT: + open disulfide bonds, − without open disulfide bonds. 5′-end modified aptamer and low salt concentration binding buffer were used here.

the cross-linking reaction and DTT to break the disulfide bond, leading to a result that only labeled histone H4 was enriched. For negative control in lane 1, in which UV light was not added and was DTT added at the same time, there was no protein captured due to the release of aptamer and no cross-linking of histone. In another control (lane 2), where neither UV light nor DTT was introduced, H4 was affinity enriched because of H4− aptamer and monoavidin−biotin interactions. In lane 4, both photo cross-linked and unlabeled histone H4 were captured since UV light was added while DTT was not, leading to a band with a deeper color than bands in lane 2 and lane 3. From the SDS-PAGE results, we can also reach the conclusion that modified aptamer and this strategy can effectively recognize and covalently label histone H4, then enrich only photolabeled target proteins under proper conditions. Previous studies suggested that the adapter might recognize the N-terminal tails of histone H4.16 However, there is no direct evidence to support this hypothesis. Here, we used 59

DOI: 10.1021/acschembio.6b00797 ACS Chem. Biol. 2017, 12, 57−62

Letters

ACS Chemical Biology

Figure 3. MS/MS identification results of histone H4 (A) before photo cross-linking as a control, and after being labeled by modified aptamer in low (B, 3′-end modified; C, 5′-end modified), middle (D, 3′-end modified; E, 5′-end modified), and high (F, 3′-end modified; G, 5′-end modified) salt binding buffer. Histone H4 structure is based on a nucleosome (PDB ID: 1KX5), and red means identified peptides; cyan, unidentified.

H4 and aptamer interface would be lower when the probe had a longer cross-linking radius because it had access to multiple regions of target protein. This prediction was supported by our MS/MS results when we compared the sequence coverages between Figure 3D and E (middle salt) and F and G (high salt); a lower identification was found in the 5′-end modified aptamers (Figure 3E and 3G) while the binding happened in middle and high salt concentration buffers. Meanwhile, it has been reported that the nucleosomes condense as the salt concentration increases and then relax at a higher salt concentration.23,24 So, it is reasonable to assume that histone H4 follows the same rule, which means it is more likely to get a higher sequence coverage when H4 is extended in low and high salt concentrations. This hypothesis was consistent with our data, the lowest sequence coverage found in middle salt concentrations (Figure 3D and E) when comparing Figure 3B, D, and F and Figure 3C, E, and G, which represented protein sequence identification in low, middle, and high salt concentrations, respectively. To prove that the missing information on the N-terminal tail is not related to inefficient digestion of certain samples or other nonconsistent experimental processes, replicate experiments were performed, and the results were provided in Figure S8. Taking all results together, we can see that the coverage significantly decreased after cross-linking and N-terminal tail identification information lost in all cross-linked samples. In other words, the decreasing sequence coverage was mainly because of cross-linking labeling. Thus, we reasonably presumed that the binding area of histone H4 to aptamer could be located at N-terminal tails. In conclusion, we designed an aptamer-based, multiple functional chemical probe and developed a novel approach for analysis of the histone H4−aptamer interaction by photo crosslinking mass spectrometry for the first time. This aptamer-based probe can be widely used under different conditions and successfully achieved a selective labeling of H4. Our results further confirmed that the binding area of histone H4 to the aptamer would be located at N-terminal tails. Thus, this strategy exhibits great potential and might further contribute to the understanding of histone−DNA interaction patterns.

aptamer-based photo cross-linking with MS identification for probing histone H4−aptamer interfaces. Because the concentration of salt has an influence on the recognition between protein and aptamer,16 three different concentrations of buffers were used for incubation of histone mixtures and aptamer probes, respectively. We also examined the efficiency for 5′-end and 3′-end modified aptamers. SDS-PAGE results (Figure S6) indicated that both kinds of modified aptamer can effectively label histone H4 no matter what kind of salt concentration was used. Next, all interesting bands were analyzed by MS. There are some difficulties in analyzing XL-MS data, and most of them could be attributed mainly to two factors. The first bottleneck is data analysis algorithms, a computational challenge known as “the n-square problem.”14 The second obstacle is insufficient fragmentation of linked peptides, which would impair the confident identification of cross-linking. For photo XL-MS, it becomes even harder because the cross-linking can happen to any amino acids, and it is challenging to get MS/ MS fragmentation after such big modification.22 Also, the photo reactive group can reach to multiple sites of target protein close enough in spatial structure due to its flexibility. Those difficulties led to a result that we could hardly find the specific binding site of protein to aptamer. Even though, we can also find clues for analyzing the histone−aptamer binding area from protein identification information. As shown in Figure 3, it was easy to find that the sequence coverage of H4 significantly decreased after photo cross-linking when compared to unlabeled histone H4. The information for the N-terminal tail could not be detected (more details can be found in Figure S7) any more. Besides the loss of N-terminal tail identification information, there were also tiny differences between different conditions, which can be explained reasonably on the basis of known structural data and flexibility of linker and protein structure changes in solution. As we mentioned before, a third stem-loop motif next to the 3′-end would be adopted and affect the aptamer−histone H4 interaction while under middle and high salt concentration conditions (Figure S1C and D). In other words, there would be a longer and more flexible linker when photoreactive groups were modified to the 5′-end of the aptamer at a higher salt concentration. Also, the resolution in deciphering the histone 60

DOI: 10.1021/acschembio.6b00797 ACS Chem. Biol. 2017, 12, 57−62

Letters

ACS Chemical Biology

■

METHODS

China with Grants (21275077), and Tianjin Municipal Science and Technology Commission (No. 14JCYBJC24000).

16

Materials. Aptamer targeting histone H4 modified with an amine group at the 5′-end or 3′-end (Apt, 5′-TGG TGG GGT TCC CGG GAG GGC GGC TAC GGG TTC CGT AAT CAG ATT TGT GT3′) was synthesized and HPLC purified by Shanghai Sangon Biological Engineering Technology. High streptavidin agarose resin, monoavidin agarose resin, and sulfo-SBED biotin label transfer reagent (SBED) were all purchased from Thermo Fisher Scientific. Dithiothreitol (DTT) and Iodoacetamide (IAA) were obtained from Sigma-Aldrich. Sequencing-grade trypsin was purchased from Promega and C18 ZipTips from Millipore. Hela cells were cultured with RPMI media 1640 supplemented with 10% FBS at 37 °C with 5% CO2 in the atmosphere. Histone proteins were acid-extracted according to the standard protocol.4 SBEDmodified aptamers were obtained by a NH2/NHS reaction between amine groups at the 5′-end or 3′-end of amino-modified DNA and a Sulfo-NHS group from the SBED agent. Aptamer-Based Affinity Enrichment of Histone H4. In brief, modified aptamers were incubated with histone proteins. In path I (Scheme 1), target proteins were eluted and determined by SDSPAGE by the use of an avidin−biotin system. While in alternative path II, photo cross-linking was initiated by UV light, and then DTT was used to open disulfide bond. Thus, only photo cross-linked proteins were enriched. Then, interesting bands were further extracted and performed in gel trypsin digestion. MS/MS Identification and Data Analysis. Samples were desalted using a μ-C18 Ziptip before HPLC-MS/MS analysis. Peptides were separated on an HPLC column with a 45 min gradient from 5 to 35% HPLC buffer B (0.1% formic acid in ACN) and identified by a QExative mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Full scan MS spectra from m/z 300−2000 were acquired in a datadependent mode, and high-energy collision dissociation energy was set at 27%. The resulting MS/MS data were searched against the human histones sequence database using the Mascot search engine. Trypsin was specified as a digesting enzyme. Acetylation, methylation, and dimethylation of lysine and methylation of arginine were searched as variable modification. A maximum of four missing cleavages were allowed. Mass tolerances for precursor ions were set at ±10 ppm for precursor ions and ±0.02 Da for MS/MS. Predicted secondary structures of aptamer in low, middle, and high salt binding buffers were calculated by using the online program OligoAnalyzer 3.1, and the PDB accession code of the histone structure is 1KX5.25

■

■

(1) Venkatesh, S., and Workman, J. L. (2015) Histone exchange, chromatin structure and the regulation of transcription. Nat. Rev. Mol. Cell Biol. 16, 178−189. (2) Su, Z., and Denu, J. M. (2015) Reading the Combinatorial Histone Language. ACS Chem. Biol. 11, 564−574. (3) Sneppen, K., and Dodd, I. B. (2012) A simple histone code opens many paths to epigenetics. PLoS Comput. Biol. 8, e1002643. (4) Shechter, D., Dormann, H. L., Allis, C. D., and Hake, S. B. (2007) Extraction, purification and analysis of histones. Nat. Protoc. 2, 1445− 1457. (5) Shao, N., Zhang, K., Chen, Y., He, X., and Zhang, Y. (2012) Preparation and characterization of DNA aptamer based spin column for enrichment and separation of histones. Chem. Commun. 48, 6684− 6686. (6) Pinto, A., Lennarz, S., Rodrigues-Correia, A., Heckel, A., O’Sullivan, C. K., and Mayer, G. n. (2011) Functional detection of proteins by caged aptamers. ACS Chem. Biol. 7, 360−366. (7) Williams, B. A., Lin, L., Lindsay, S. M., and Chaput, J. C. (2009) Evolution of a histone H4-K16 acetyl-specific DNA aptamer. J. Am. Chem. Soc. 131, 6330−6331. (8) Price, B. D., and D’Andrea, A. D. (2013) Chromatin remodeling at DNA double-strand breaks. Cell 152, 1344−1354. (9) Dey, B., Thukral, S., Krishnan, S., Chakrobarty, M., Gupta, S., Manghani, C., and Rani, V. (2012) DNA-protein interactions: methods for detection and analysis. Mol. Cell. Biochem. 365, 279−299. (10) Zhang, Q., Chen, J., Kuwajima, K., Zhang, H.-M., Xian, F., Young, N. L., and Marshall, A. G. (2013) Nucleotide-induced conformational changes of tetradecameric GroEL mapped by H/D exchange monitored by FT-ICR mass spectrometry, Sci. Rep., 3, DOI: 10.1038/srep01247. (11) Weng, L., Zhou, C., and Greenberg, M. M. (2014) Probing interactions between lysine residues in histone tails and nucleosomal DNA via product and kinetic analysis. ACS Chem. Biol. 10, 622−630. (12) DeNizio, J. E., Elsässer, S. J., and Black, B. E. (2014) DAXX cofolds with H3. 3/H4 using high local stability conferred by the H3. 3 variant recognition residues. Nucleic Acids Res. 42, 4318−4331. (13) Liu, F., Rijkers, D. T., Post, H., and Heck, A. J. (2015) Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. Nat. Methods 12, 1179−1184. (14) Liu, F., and Heck, A. J. (2015) Interrogating the architecture of protein assemblies and protein interaction networks by cross-linking mass spectrometry. Curr. Opin. Struct. Biol. 35, 100−108. (15) Deroo, S. p., Stengel, F., Mohammadi, A., Henry, N., Hubin, E., Krammer, E.-M., Aebersold, R., and Raussens, V. (2015) Chemical cross-linking/mass spectrometry maps the amyloid β peptide binding region on both apolipoprotein E domains. ACS Chem. Biol. 10, 1010− 1016. (16) Yu, H., Jiang, B., and Chaput, J. C. (2011) Aptamers can discriminate alkaline proteins with high specificity. ChemBioChem 12, 2659−2666. (17) Dundas, C. M., Demonte, D., and Park, S. (2013) Streptavidinbiotin technology: improvements and innovations in chemical and biological applications. Appl. Microbiol. Biotechnol. 97, 9343−9353. (18) Wu, S. C., and Wong, S. L. (2005) Engineering soluble monomeric streptavidin with reversible biotin binding capability. J. Biol. Chem. 280, 23225−23231. (19) Huguet, J. M., Bizarro, C. V., Forns, N., Smith, S. B., Bustamante, C., and Ritort, F. (2010) Single-molecule derivation of salt dependent base-pair free energies in DNA. Proc. Natl. Acad. Sci. U. S. A. 107, 15431−15436. (20) Howell, L. A., Waller, Z. A., Bowater, R., O’Connell, M., and Searcey, M. (2011) A small molecule that induces assembly of a four way DNA junction at low temperature. Chem. Commun. 47, 8262− 8264.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00797. Experimental procedures in detail, SDS-PGAE results, and mass spectrometry data (PDF)

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kai Zhang: 0000-0003-2800-0531 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (Grants 2013CB910903, 2016YFC0903000, and 2012CB910601), National Natural Science Foundation of 61

DOI: 10.1021/acschembio.6b00797 ACS Chem. Biol. 2017, 12, 57−62

Letters

ACS Chemical Biology (21) Balamurugan, S., Obubuafo, A., McCarley, R. L., Soper, S. A., and Spivak, D. A. (2008) Effect of linker structure on surface density of aptamer monolayers and their corresponding protein binding efficiency. Anal. Chem. 80, 9630−9634. (22) Sinz, A. (2006) Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein−protein interactions. Mass Spectrom. Rev. 25, 663−682. (23) Sahasrabuddhe, C., and Saunders, G. F. (1977) Salt-induced structural changes in nucleosomes. Nucleic Acids Res. 4, 853−866. (24) Dieterich, A. E., Axel, R., and Cantor, C. R. (1979) Salt-induced structural changes of nucleosome core particles. J. Mol. Biol. 129, 587− 602. (25) Davey, C. A., Sargent, D. F., Luger, K., Maeder, A. W., and Richmond, T. J. (2002) Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution. J. Mol. Biol. 319, 1097−1113.

62

DOI: 10.1021/acschembio.6b00797 ACS Chem. Biol. 2017, 12, 57−62