Revealing transient structures of nucleosomes as DNA unwinds.
Bottom Line: The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications.We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly.These kinetic intermediates may be biologically important substrates for gene regulation.
Affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.Show MeSH
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Mentions: Small angle X-ray scattering (SAXS) is a label-free technique that reports the global conformation and composition of macromolecules, including NCPs, in solution (21–26). The scattered intensity provides information about the average composition, size and shape of the scattering particles. The extrapolated scattering intensity at zero angle, I(0), is proportional to the square of the excess electron density of the particles in solution and is therefore sensitive to changes in the oligomeric state of the complexes. Thus, I(0) can be used to monitor the dissociation of proteins from the NCP. A quantitative measure of size is reported as the radius of gyration (Rg). For scatterers with homogenous electron densities, the scattered intensity I(q) is directly related to macromolecular shape. However, for complexes with components that have varying electron densities (e.g. protein and nucleic acids), the relationship between I(q) and macromolecular shape becomes ambiguous. The simplest way to circumvent this challenge is to apply contrast variation and match the electron density of solvent with the lower density protein (see Supplementary Text: Contrast Variation). By adding 50% sucrose to the solvent, the protein becomes invisible above the background and only the DNA contributes to the scattering (Figure 1). Contrast variation SAXS has successfully revealed the structure of RNA or DNA complexed with proteins in static studies (27–28). Here we describe the application of contrast variation to monitor changing NCP conformations as [NaCl] is increased in equilibrium titrations. We then expand on this strategy by incorporating a stopped-flow mixer (SFM) to measure time-dependent changes following the rapid addition of salt (Figure 2). Extensive characterization of mixing performance verified a ≈ 5 ms mixing dead time, even for viscous sucrose solutions (see Supplementary Text: Mixer Characterization).
Affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.