A genetically encoded anomalous SAXS ruler to probe the dimensions of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) adopt ensembles of rapidly fluctuating heterogeneous conformations, influencing their binding capabilities and supramolecular transitions. The primary conformational descriptors for understanding IDP ensembles-the radius of gyration (), measured by small-angle X-ray scattering (SAXS), and the root mean square (rms) end-to-end distance (), probed by fluorescent resonance energy transfer (FRET)-are often reported to produce inconsistent results regarding IDP expansion as a function of denaturant concentration in the buffer. This ongoing debate surrounding the FRET-SAXS discrepancy raises questions about the overall reliability of either method for quantitatively studying IDP properties. To address this discrepancy, we introduce a genetically encoded anomalous SAXS (ASAXS) ruler, enabling simultaneous and direct measurements of and without assuming a specific structural model. This ruler utilizes a genetically encoded noncanonical amino acid with two bromine atoms, providing an anomalous X-ray scattering signal for precise distance measurements. Through this approach, we experimentally demonstrate that the ratio between and varies under different denaturing conditions, highlighting the intrinsic properties of IDPs as the primary source of the observed SAXS-FRET discrepancy rather than shortcomings in either of the two established methods. The developed genetically encoded ASAXS ruler emerges as a versatile tool for both IDPs and folded proteins, providing a unified approach for obtaining complementary and site-specific conformational information in scattering experiments, thereby contributing to a deeper understanding of protein functions.