Contrast variation SAXS: Sample preparation protocols, experimental procedures, and data analysis.

Abstract

Proteins and nucleic acids, alone and in complex are among the essential building blocks of living organisms. Obtaining a molecular level understanding of their structures, and the changes that occur as they interact, is critical for expanding our knowledge of life processes or disease progression. Here, we motivate and describe an application of solution small angle X-ray scattering (SAXS) which provides valuable information about the structures, ensembles, compositions and dynamics of protein-nucleic acid complexes in solution, in equilibrium and time-resolved studies. Contrast variation (CV-) SAXS permits the visualization of the distinct molecular constituents (protein and/or nucleic acid) within a complex. CV-SAXS can be implemented in two modes. In the simplest, the protein within the complex is effectively rendered invisible by the addition of an inert contrast agent at an appropriate concentration. Under these conditions, the structure, or structural changes of only the nucleic acid component of the complex can be studied in detail. The second mode permits observation of both components of the complex: the protein and the nucleic acid. This approach requires the acquisition of SAXS profiles on the complex at different concentrations of a contrast agent. Here, we review CV-SAXS as applied to protein-nucleic acid complexes in both modes. We provide some theoretical framework for CV-SAXS but focus primarily on providing the necessary information required to implement a successful experiment including experimental design, sample quality assessment, and data analysis.