Molecular and atomic level characterization of transcription factor (TF)-DNA complexes is critical for understanding DNA-binding specificity and potentially structural changes that may occur in protein and/or DNA upon complex formation. Often TFs are large, multidomain proteins or contain disordered regions that contribute to DNA recognition and/or binding affinity but are difficult to structurally characterize due to their high molecular weight and intrinsic flexibility. This results in challenges to obtaining high resolution structural information using Nuclear Magnetic Resonance (NMR) spectroscopy due to the relatively large size of the protein-DNA complexes of interest or macromolecular crystallography due to the difficulty in obtaining crystals of flexible proteins. Small angle X-ray scattering (SAXS) offers a complementary method to NMR and X-ray crystallography that allows for low-resolution structural characterization of protein, DNA, and protein-DNA complexes in solution over a greater size range and irrespective of interdomain flexibility and/or disordered regions. One important caveat to SAXS data interpretation, however, has been the inability to distinguish between scattering coming from the protein versus DNA component of the complex of interest. Here, we present a protocol using contrast variation via increasing sucrose concentrations to distinguish between protein and DNA using the model protein bovine serum albumin (BSA) and DNA and the LUX ARRYTHMO TF-DNA complex. Examination of the scattering curves of the components individually and in combination with contrast variation allows the differentiation of protein and DNA density in the derived models. This protocol is designed for use on high flux SAXS beamlines with temperature-controlled sample storage and sample exposure units.
Keywords: Contrast variation; Protein-DNA complexes; Small angle X-ray scattering; Transcription factors.
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