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

le Paige, Ulric B.; Xiang, ShengQi; Hendrix, Marco M. R. M.; Zhang, Yi; Folkers, Gert E.; Weingarth, Markus; Bonvin, Alexandre M. J. J.; Kutateladze, Tatiana G.; Voets, Ilja K.; Baldus, Marc; van Ingen, Hugo

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 inline-formula∼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.



le Paige, Ulric B. / Xiang, ShengQi / Hendrix, Marco M. R. M. / et al: Characterization of nucleosome sediments for protein interaction studies by solid-state NMR spectroscopy. 2021. Copernicus Publications.


12 Monate:

Grafik öffnen


Rechteinhaber: Ulric B. le Paige et al.

Nutzung und Vervielfältigung: