Abstract
Regulation of DNA-templated processes such as gene transcription and DNA repair depend on the interaction of a wide range of proteins with the nucleosome, the fundamental building block of chromatin. Both solution and solid-state NMR spectroscopy have become an attractive approach to study the dynamics and interactions of nucleosomes, despite their high molecular weight of ~ 200 kDa. For solid-state NMR (ssNMR) studies, dilute solutions of nucleosomes are converted to a dense phase by sedimentation or precipitation. Since nucleosomes are known to self-associate, these dense phases may induce extensive interactions between nucleosomes, which could interfere with protein-binding studies. Here, we characterized the packing of nucleosomes in the dense phase created by sedimentation using NMR and small-angle X-ray scattering (SAXS) experiments. We found that nucleosome sediments are gels with variable degrees of solidity, have nucleosome concentration close to that found in crystals, and are stable for weeks under high-speed magic angle spinning (MAS). Furthermore, SAXS data recorded on recovered sediments indicate that there is no pronounced long-range ordering of nucleosomes in the sediment. Finally, we show that the sedimentation approach can also be used to study low-affinity protein interactions with the nucleosome. Together, our results give new insights into the sample characteristics of nucleosome sediments for ssNMR studies and illustrate the broad applicability of sedimentation-based NMR studies.
Highlights
Both prokaryotes and eukaryotes use an advanced protein machinery to regulate the expression and maintenance of their genome
To assess the impact on the study of nucleosome–protein interactions, we focused on the second PHD finger of CHD4 as a test case
As a first characterization of the nucleosome sediment in the solid-state NMR (ssNMR) rotor, we assessed the nucleosome concentration for four different sample preparations from absorbance measurements of the solution before and after ultracentrifugation
Summary
Both prokaryotes and eukaryotes use an advanced protein machinery to regulate the expression and maintenance of their genome. Determining the molecular basis of the underlying interactions is crucial for our fundamental understanding of biology and for developing new treatments for disease. The regulatory proteins have direct access to the DNA. Ground-breaking NMR studies made a major contribution to our understanding of how such proteins search and recognize their target DNA sequences (Boelens et al, 1987; Spronk et al, 1999; Kalodimos et al, 2001, 2004). The DNA is packaged in nucleosomes, a protein–DNA complex formed by ∼ 145–147 bp of DNA that are wrapped around a core of histone proteins (Fig. 1a). The histones H2A, H2B, H3 and H4 form an octameric complex that binds the DNA.
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