Preprints
https://doi.org/10.5194/mr-2021-21
https://doi.org/10.5194/mr-2021-21

  19 Feb 2021

19 Feb 2021

Review status: this preprint is currently under review for the journal MR.

Characterization of nucleosome sediments for protein interaction studies by solid-state NMR spectroscopy

Ulric B. le Paige1, ShengQi Xiang1,a, Marco M. R. M. Hendrix2, Yi Zhang3, Markus Weingarth1, Alexandre M. J. J. Bonvin1, Tatiana G. Kutateladze3, Ilja K. Voets2, Marc Baldus1, and Hugo van Ingen1 Ulric B. le Paige et al.
  • 1Utrecht NMR Group, Bijvoet Centre for Biomolecular Research, Utrecht University, 3584 CH, Utrecht, The Netherlands
  • 2Laboratory of Self-Organizing Soft Matter, Department of Chemical Engineering and Chemistry & Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven, The Netherlands
  • 3Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA
  • acurrent address: MOE Key Lab for Membrane-less Organelles & Cellular Dynamics, School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, 230026, Anhui, China

Abstract. Regulation of DNA-templated processes such as gene transcription and DNA repair depend on the interaction of a wide range of proteins to 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.

Ulric B. le Paige et al.

Status: open (until 19 Mar 2021)

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Ulric B. le Paige et al.

Ulric B. le Paige et al.

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Short summary
NMR studies can be of great help to understand the molecular mechanisms of nucleosome function. For solid-state NMR, nucleosomes need to be tightly packed together. We show that centrifugation of nucleosomes results in formation of gels with very high packing ratios yet without pronounced order in the packing and without formation of specific or stable inter-nucleosome contacts. This makes the approach suitable also for the study of proteins that bind weakly to the nucleosome.