Despite their biological importance, few methods exist to monitor these complexes as they assemble, dissociate or function. The ability to observe these dynamics in action will present new strategies for manipulating gene expression.
Using CHESS G-line x-rays, the Pollack group (Cornell, Applied Physics), in collaboration with the Gloss lab (Washington State University, Molecular Biosciences) recently demonstrated a new experimental method for tracking the real time motions of DNA as it unwinds from a protein (histone) core in a nucleosome core particle (NCP), the fundamental repeating unit of DNA packaging in chromatin. Transient structures are revealed as DNA is released from this tight storage structure.
This new method combines time-resolved small angle x-ray scattering (SAXS), that reveals the dynamic size and shape of macromolecules, with contrast variation to highlight scattering only from the DNA component in a dynamically changing protein-DNA complex. The contrast variation method (Figure 1) involves adding sucrose to increase the electron density of the surrounding solution until it exactly matches that of the lighter, protein component. Under this contrast matched condition, the protein scatter ‘disappears’ and only the DNA is visible above the background. Contrast matching is frequently employed in neutron scattering experiments, where the contrast of the surrounding solution can be varied over a wide range using mixtures of D2O and H2O. However, the high flux of synchrotron x-rays is essential for rapid, time-resolved experiments.
To promote DNA unwrapping, NCPs in sucrose are rapidly mixed with salt in a stopped flow mixer. After mixing, the sample is injected into an x-ray compatible capillary for use at G1. SAXS profiles, acquired with time resolution as sharp as 20 milliseconds, reveal the changing DNA structures shown schematically in Figure 2.
SAXS experiments without sucrose report the degree of protein association to the DNA (Figure 2, 0% sucrose panel). When sucrose is added, only the DNA structures contribute to the scattering profile (Figure 2, 50% sucrose panel). A transiently populated intermediate is detected, with a lifetime of 200 ms. This short-lived state displays asymmetric DNA release from a disrupted, but still attached histone core. No other method provides such complete information about the composition and structures of transiently populated states.
Time-resolved, contrast variation SAXS can be extended to follow global structural changes of any protein nucleic acid complex that can be initiated by a mixing reaction. Macromolecular machines including the ribosome or the spliceosome are potential targets for this powerful new technology.
A paper describing this work, with lead authors Yujie Chen and Joshua Tokuda, recently appeared in Nucleic Acids Research.
References:
[1] Chen Y, Tokuda J, Topping T, Sutton JL, Meisburger SP, Pabit SA, Gloss LM and Pollack L, "Revealing transient structures of nucleosomes as DNA unwinds", Nucleic Acids Research 42(13), 8767-8776 (2014).
See also: http://phys.org/news/2014-08-dna-unwrap-blank-proteins.html