Histone H3 and H4 N-Terminal Tails in Nucleosome Arrays at Cellular Concentrations Probed by Magic Angle Spinning NMR Spectroscopy

Abstract

Chromatin is a supramolecular assembly of DNA and histone proteins, organized into nucleosome repeat units. The dynamics of chromatin organization regulates DNA accessibility to eukaryotic transcription and DNA repair complexes. However, the structural and dynamic properties of chromatin at high concentrations characteristic of the cellular environment (> ~200 mg/ml) are largely unexplored at the molecular level. Here, we apply magic angle spinning nuclear magnetic resonance to directly probe the dynamic histone protein regions in 13C,15N-enriched recombinant nucleosome arrays at cellular chromatin concentrations and conditions designed to emulate distinct states of DNA condensation, with focus on the flexible H3 and H4 N-terminal tails which mediate chromatin compaction. 2D 1H-13C and 1H-15N spectra reveal numerous correlations for H3 and H4 backbone and side-chain atoms, enabling identification of specific residues making up the dynamically disordered N-terminal tail domains. Remarkably, we find that both the H3 and H4 N-terminal tails are overall dynamic even in a highly condensed state. This significant conformational flexibility of the histone tails suggests that they remain available for protein binding in compact chromatin states to enable regulation of heterochromatin. Furthermore, our study provides a foundation for quantitative structural and dynamic investigations of chromatin at physiological concentrations.

Coupling between Histone Conformations and DNA Geometry in Nucleosomes on a Microsecond Timescale: Atomistic Insights into Nucleosome Functions

“…For instance, the amplitude of fluctuations of the H3 tail on one side of nucleosome, which adsorbed on the linker DNA, was much higher (due to linker DNA flexibility) compared to the other H3 tail, which was compactly folded at the DNA entry/exit sites. The dynamical scales of histone tails in mononucleosomes and nucleosomal arrays were recently probed by a number of techniques including solution, solid state NMR and hydrogen exchange studies [31–33]. They reported rather controversial findings.…”

“…The microsecond timescale as well as nucleosome models with the realistic DNA linker segments and multiple comparative simulations enabled us to get insights into the functionally relevant rearrangements in nucleosome including the coupling between the conformations of histone tails and the DNA geometry. While many functionally relevant motions may occur on much longer time scales [10], several NMR studies recently suggested that histone tails showed sub-microsecond dynamics providing possibility to compare our computational results with the experimental data [31–33]. …”

Site‐Specific Studies of Nucleosome Interactions by Solid‐State NMR Spectroscopy

“…Sedimentation has been widely used to study the folding and compaction of nucleosomes and nucleosomal arrays 5. Jaroniec and co‐workers were the first to realize the great potential of ssNMR spectroscopy in the context of nucleosomes through their study on histone tails in nucleosomal arrays 6. Herein, we introduce the use of nucleosome sedimentation, ultra‐fast magic angle spinning (MAS), and 1 H‐detected ssNMR spectroscopy to characterize the structure and dynamics of nucleosomes and their protein complexes.…”

High Accuracy Protein Structures from Minimal Sparse Paramagnetic Solid‐State NMR Restraints

“…[1][2][3][4][5] In spite of the latest advances, [6,7] high-throughput protein structure elucidation by conventional ssNMR methods remains challenging due to difficulties in obtaining critical non-local contacts.D uring the past decade,s sNMR measurements of nuclear paramagnetic relaxation enhancements (PREs) have been explored for structural studies of native metalloproteins [8,9] and proteins modified with covalent paramagnetic tags,including nitroxide spin labels [10] and metal chelates. [1][2][3][4][5] In spite of the latest advances, [6,7] high-throughput protein structure elucidation by conventional ssNMR methods remains challenging due to difficulties in obtaining critical non-local contacts.D uring the past decade,s sNMR measurements of nuclear paramagnetic relaxation enhancements (PREs) have been explored for structural studies of native metalloproteins [8,9] and proteins modified with covalent paramagnetic tags,including nitroxide spin labels [10] and metal chelates.…”

“…Magic-angle spinning solid-state nuclear magnetic resonance (ssNMR) has emerged as av iable tool for analysis of the structure and dynamics of biomacromolecular complexes and assemblies including membrane proteins,a myloids,v iral capsids,a nd chromatin. [1][2][3][4][5] In spite of the latest advances, [6,7] high-throughput protein structure elucidation by conventional ssNMR methods remains challenging due to difficulties in obtaining critical non-local contacts.D uring the past decade,s sNMR measurements of nuclear paramagnetic relaxation enhancements (PREs) have been explored for structural studies of native metalloproteins [8,9] and proteins modified with covalent paramagnetic tags,including nitroxide spin labels [10] and metal chelates. [11][12][13] These paramagnetic methods are capable of providing information about electronnucleus distances up to~20 ,p roviding valuable information for structure determination.…”