Characterizing flexible macromolecules with SAXS.



Unstructured proteins, RNA or DNA components provide functionally important flexibility that is key to many macromolecular assemblies throughout cell biology. As objective, quantitative experimental measures of flexibility and disorder in solution are limited, small angle scattering (SAS), and in particular small angle X-ray scattering (SAXS), provides a critical technology to assess macromolecular flexibility as well as shape and assembly. In a recent paper published in Biopolymers, Rob Rambo and John Tainer consider the Porod-Debye law as a powerful tool for detecting biopolymer flexibility in SAS experiments. They show that the Porod-Debye region fundamentally describes the nature of the scattering intensity decay, which captures information needed for distinguishing between folded and flexible particles. Particularly for comparative SAS experiments, application of the law, as described in their manuscript, can distinguish between discrete conformational changes and localized flexibility relevant to molecular recognition and interaction networks. This approach aids insightful analyses of fully and partly flexible macromolecules that is more robust and conclusive than traditional Kratky analyses. Furthermore, they demonstrate for prototypic SAXS data that the ability to calculate particle density by the Porod-Debye criteria provides an objective quality assurance parameter that may prove of general use for SAXS modeling and validation.

The figure, excerpted from the manuscript, shows SAXS experiments performed on an exemplary intrinsically disordered domain Rad51 AP1. Data was collected for both rad51 AP1 (red) and a fusion construct with E. coli maltose binding protein (MBP) (black). (A) Kratky plot demonstrating the parabolic shape for a partially folded particle (black) and hyperbolic shape for a full unfolded particle (red). (B) Porod-Debye plot demonstrating a clear plateau for the partially folded MBP-Rad51 AP1 hybrid particle. In the absence of MBP, the fully unfolded domain is devoid of any discernible plateau.

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