A genetically encoded ASAXS ruler to study the dimension of intrinsically disordered proteins
Final Report Abstract
Intrinsically disordered proteins (IDPs) lack stable secondary or quaternary structures, instead forming dynamic ensembles of interchanging conformations. A key property of these ensem-bles is their scaling behavior, which describes how their dimensions grow with the number of residues. Polymer theory links the scaling exponent to dominant molecular interactions—whether self-self or self-solvent interactions prevail. Biologically, scaling behavior helps predict protein functions, including their involvement in phase separation and aggregation pro-cesses in general. Common experimental techniques to study these conformations include the radius of gyration (RG) from small-angle X-ray scattering (SAXS) and the end-to-end distance (RE) from fluorescence resonance energy transfer spectroscopy (FRET), particularly single-molecule FRET (smFRET). However, persistent disagreement between SAXS and FRET results has sparked significant debate, highlighted by conflicting high-profile publications. This discrepancy about the fundamental parameter on protein structure and function slows down scientific progress in the field of protein aggregation. To address this discrepancy, we introduced a genetically encoded anomalous SAXS (ASAXS) ruler, enabling simultaneous and direct measurements of RG and RE without assuming a specific structural model. This ruler utilises a novel self-synthesised noncanonical amino acid (ncAA) with two bromine atoms that was subsequentially genetically encoded using Amber suppression technology. This ncAA provides an anomalous X-ray scattering signal for precise distance measurements. Through this approach, we experimentally demonstrate that the ratio between RG and RE 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.
Publications
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A genetically encoded anomalous SAXS ruler to probe the dimensions of intrinsically disordered proteins. Proceedings of the National Academy of Sciences, 121(50).
Yu, Miao; Gruzinov, Andrey Yu.; Ruan, Hao; Scheidt, Tom; Chowdhury, Aritra; Giofrè, Sabrina; Mohammed, Ahmed S. A.; Caria, Joana; Sauter, Paul F.; Svergun, Dmitri I. & Lemke, Edward A.
