The Nucleosome Revisited: Combined Small-Angle X-Ray/Neutron Scattering and Molecular Dynamics Applied to Reconstituted Mononucleosomes

Abstract

Nucleosomes are the fundamental structural units of chromatin. A mononucleosome is comprised of 147 base pairs of DNA wrapped around a heterooctameric core of histone proteins that include H2A, H2B, H3, and H4, and other naturally occurring functional variants. In the mid-1970s, the SANS technique was key to the determination of the composition, structure, and conformation of the DNA within this basic unit, fifteen years before the first x-ray diffraction results were available. Small-angle scattering is a long-established technique that is well-suited for the study of macromolecules in vitro, as they exist in solution. Small-angle neutron scattering (SANS) is ideally suited to study nucleoprotein complexes, since the neutron scattering strengths of the different macromolecules differ with respect to each other. By employing contrast variation techniques, where the D2O:H2O ratio of the solutions is systematically varied, the gross structure and distribution of the individual components of a composite particle can be resolved in a model-independent fashion.We will present work which revisits these decades-old canonical measurements with a modern perspective. Experimental neutrons from beam line CG-3 at the ORNL High-Flux Isotope Reactor (HFIR) were successfully utilized to study reconstituted mononucleosomes by contrast variation. Our work marries this method with analytical centrifugation and the atomistic modeling approaches implemented in the program SASSIE. This approach allows us to directly reconcile available atomic structures with their solution properties, resulting in complete single and ensemble solution models. As a result of this work, we will present new insights into the distribution of the protein and DNA components of the mononucleosome in solution, including the properties of histone tails in solution and DNA wrapping. The methods presented provide an experimental framework for future examination of variant nucleosome and nucleosome-derived higher order assemblies.