scholarly journals “Dual protease type XIII/pepsin digestion offers superior resolution and overlap for the analysis of histone tails by HX-MS”

2019 ◽  
Author(s):  
James Mullahoo ◽  
Terry Zhang ◽  
Karl Clauser ◽  
Steven A. Carr ◽  
Jacob D. Jaffe ◽  
...  
Keyword(s):  
Hydrogen Exchange ◽  
Electron Transfer Dissociation ◽  
Histone Tails ◽  
Post Translational Modifications ◽  
Pepsin Digestion ◽  
Exchange Mass Spectrometry ◽  
Nucleosome Core ◽  
Nucleosome Core Particles ◽  
Hydrogen Deuterium ◽  
Dynamic Structures

AbstractThe N-terminal regions of histone proteins (tails) are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications (PTMs) present on these regions contribute to transcriptional regulation. While hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed to analyze histones due to the poor N-terminal coverage obtained using pepsin. Here, we test the applicability of a dual protease type XIII/pepsin digestion column to this class of proteins. We optimize online digestion conditions using the H4 monomer, and extend the method to the analysis of histones in monomeric states and nucleosome core particles (NCPs). We show that the dual protease column generates many short and overlapping N-terminal peptides. We evaluate our method by performing hydrogen exchange experiments of NCPs for different time points and present full coverage of the tails at excellent resolution. We further employ electron transfer dissociation (ETD) and showcase an unprecedented degree of overlap across multiple peptides that is several fold higher than previously reported methods. The method we report here may be readily applied to the HX-MS investigation of histone dynamics and to the footprints of histone binding proteins on nucleosomes.

Role of Histone Tails in the Conformation and Interactions of Nucleosome Core Particles†

Biochemistry ◽  
2004 ◽  
Vol 43 (16) ◽  
pp. 4773-4780 ◽  
Author(s):  
Aurélie Bertin ◽  
Amélie Leforestier ◽  
Dominique Durand ◽  
Françoise Livolant
Keyword(s):  
Histone Tails ◽  
Nucleosome Core ◽  
Nucleosome Core Particles ◽  
Core Particles

scholarly journals Hydrogen-deuterium exchange coupled to top- and middle-down mass spectrometry enables high-resolution measurements of histone tail dynamics before and after nucleosome assembly

2018 ◽  
Author(s):  
Kelly R. Karch ◽  
Mariel Coradin ◽  
Levani Zandarashvili ◽  
Zhong-Yuan Kan ◽  
Morgan Gerace ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer Dissociation ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Nucleosome Assembly ◽  
Histone Tail ◽  
Deuterium Label ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Hdx Ms

AbstractUntil recently, a major limitation of hydrogen deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization information was limited to the length of the peptide generated during proteolysis. Recently, however, it has been demonstrated that electron transfer dissociation (ETD) allows for preservation of deuterium label in the gas phase and therefore can be used to obtain more resolved information. To date, this technology has remained mostly limited to single, small, already well-characterized model proteins. Here, we optimize, expand, and adapt HDX-MS/MS capabilities to accommodate histone and nucleosomal complexes on top-down (TD) HDX-MS/MS and middle-down (MD) HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded rigorous analysis by traditional structural biology techniques for decades, revealing important novel insights into chromatin biology. This work represents the first heterogeneous protein complex and protein-DNA complex to be analyzed by TD- and MD-HDX-MS/MS, respectively. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.

scholarly journals Dynamic networks observed in the nucleosome core particles couple the histone globular domains with DNA

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Xiangyan Shi ◽  
Chinmayi Prasanna ◽  
Aghil Soman ◽  
Konstantin Pervushin ◽  
Lars Nordenskiöld
Keyword(s):  
Dynamic Networks ◽  
Structural Features ◽  
Epigenetic Changes ◽  
Internal Dynamics ◽  
Histone Tails ◽  
Multiple Timescales ◽  
Nucleosome Core ◽  
The Core ◽  
Nucleosome Core Particles ◽  
Core Particles

Abstract The dynamics of eukaryotic nucleosomes are essential in gene activity and well regulated by various factors. Here, we elucidated the internal dynamics at multiple timescales for the human histones hH3 and hH4 in the Widom 601 nucleosome core particles (NCP), suggesting that four dynamic networks are formed by the residues exhibiting larger-scale μs-ms motions that extend from the NCP core to the histone tails and DNA. Furthermore, despite possessing highly conserved structural features, histones in the telomeric NCP exhibit enhanced μs-ms dynamics in the globular sites residing at the identified dynamic networks and in a neighboring region. In addition, higher mobility was observed for the N-terminal tails of hH3 and hH4 in the telomeric NCP. The results demonstrate the existence of dynamic networks in nucleosomes, through which the center of the core regions could interactively communicate with histone tails and DNA to potentially propagate epigenetic changes.

scholarly journals Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry

2017 ◽  
Vol 474 (11) ◽  
pp. 1867-1877 ◽  
Author(s):  
Glenn R. Masson ◽  
Sarah L. Maslen ◽  
Roger L. Williams
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer ◽  
Electron Transfer Dissociation ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Phosphoinositide 3 Kinase ◽  
Hdx Ms

Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics.

scholarly journals Histone tails decrease N7-methyl-2′-deoxyguanosine depurination and yield DNA–protein cross-links in nucleosome core particles and cells

2018 ◽  
Vol 115 (48) ◽  
pp. E11212-E11220 ◽  
Author(s):  
Kun Yang ◽  
Daeyoon Park ◽  
Natalia Y. Tretyakova ◽  
Marc M. Greenberg
Keyword(s):  
Alkylating Agents ◽  
Histone Variants ◽  
Site Formation ◽  
Abasic Site ◽  
Histone Tails ◽  
Nucleosome Core ◽  
Nucleosome Core Particles ◽  
Cross Links ◽  
Positively Charged ◽  
Core Particles

Monofunctional alkylating agents preferentially react at the N7 position of 2′-deoxyguanosine in duplex DNA. Methylated DNA, such as that produced by methyl methanesulfonate (MMS) and temozolomide, exists for days in organisms. The predominant consequence of N7-methyl-2′-deoxyguanosine (MdG) is widely believed to be abasic site (AP) formation via hydrolysis, a process that is slow in free DNA. Examination of MdG reactivity within nucleosome core particles (NCPs) provided two general observations. MdG depurination rate constants are reduced in NCPs compared with when the identical DNA sequence is free in solution. The magnitude of the decrease correlates with proximity to the positively charged histone tails, and experiments in NCPs containing histone variants reveal that positively charged amino acids are responsible for the decreased rate of abasic site formation from MdG. In addition, the lysine-rich histone tails form DNA–protein cross-links (DPCs) with MdG. Cross-link formation is reversible and is ascribed to nucleophilic attack at the C8 position of MdG. DPC and retarded abasic site formation are observed in NCPs randomly damaged by MMS, indicating that these are general processes. Histone–MdG cross-links were also detected by mass spectrometry in chromatin isolated from V79 Chinese hamster lung cells treated with MMS. The formation of DPCs following damage by a monofunctional alkylating agent has not been reported previously. These observations reveal the possibility that such DPCs may contribute to the cytotoxicity of monofunctional alkylating agents, such as MMS, N-methyl-N-nitrosourea, and temozolomide.

PROTEIN DYNAMICS IN PHOSPHORYLATION-MEDIATED ALLOSTERY PROBED BY AMIDE EXCHANGE MASS SPECTROMETRY

COSMOS ◽  
2013 ◽  
Vol 09 (01) ◽  
pp. 19-27
Author(s):  
MADHUBRATA GHOSH ◽  
GANESH S. ANAND
Keyword(s):  
Mass Spectrometry ◽  
Protein Dynamics ◽  
Protein Function ◽  
Post Translational Modifications ◽  
Exchange Mass Spectrometry ◽  
Amide Exchange ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
And Function

A major goal of molecular biology is to correlate molecular structure with function. Since most enzymes and biological catalysts are proteins, the focus for correlating 'form' with 'function' has been entirely on protein macromolecular structure. It is obvious that any understanding of protein function must come through an understanding protein dynamics. Furthermore, all of the regulatory reactions are through changes in dynamics brought about by post-translational modifications, the most important of which is phosphorylation. This review highlights the important role of covalent phosphorylation and noncovalent phosphates in regulating allosteric effects and function through a study of protein dynamics. Mass spectrometry is a relatively new and increasingly important tool for describing protein dynamics. All examples described in this review have been studied by amide hydrogen/deuterium exchange mass spectrometry.

scholarly journals The Influence of Ionic Environment and Histone Tails on Columnar Order of Nucleosome Core Particles

2016 ◽  
Vol 110 (8) ◽  
pp. 1720-1731 ◽  
Author(s):  
Nikolay V. Berezhnoy ◽  
Ying Liu ◽  
Abdollah Allahverdi ◽  
Renliang Yang ◽  
Chun-Jen Su ◽  
...  
Keyword(s):  
Histone Tails ◽  
Nucleosome Core ◽  
Ionic Environment ◽  
Nucleosome Core Particles ◽  
Core Particles

scholarly journals Conformational Change of Tetratricopeptide Repeats Region Triggers Activation of Phytochrome-Associated Protein Phosphatase 5

2021 ◽  
Vol 12 ◽  
Author(s):  
Silke von Horsten ◽  
Lars-Oliver Essen
Keyword(s):  
Conformational Changes ◽  
Protein Phosphatase ◽  
Bound State ◽  
Cd Spectroscopy ◽  
Tetratricopeptide Repeats ◽  
Post Translational Modifications ◽  
Protein Phosphatase 5 ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Dynamics Analyses

Phytochrome activity is not only controlled by light but also by post-translational modifications, e. g. phosphorylation. One of the phosphatases responsible for plant phytochrome dephosphorylation and thereby increased activity is the phytochrome-associated protein phosphatase 5 (PAPP5). We show that PAPP5 recognizes phospho-site mimicking mutants of phytochrome B, when being activated by arachidonic acid (AA). Addition of AA to PAPP5 decreases the α-helical content as tracked by CD-spectroscopy. These changes correspond to conformational changes of the regulatory tetratricopeptide repeats (TPR) region as shown by mapping data from hydrogen deuterium exchange mass spectrometry onto a 3.0 Å crystal structure of PAPP5. Surprisingly, parts of the linker between the TPR and PP2A domains and of the so-called C-terminal inhibitory motif exhibit reduced deuterium uptake upon AA-binding. Molecular dynamics analyses of PAPP5 complexed to a phyB phosphopeptide show that this C-terminal motif remains associated with the TPR region in the substrate bound state, suggesting that this motif merely serves for restricting the orientations of the TPR region relative to the catalytic PP2A domain. Given the high similarity to mammalian PP5 these data from a plant ortholog show that the activation mode of these PPP-type protein phosphatases is highly conserved.

Sign in / Sign up

Export Citation Format

Share Document