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

• 成果类型: Article
• 署名作者: Yu, Miao; Gruzinov, Andrey Yu.; Ruan, Hao; Scheidt, Tom; Chowdhury, Aritra; Giofre, Sabrina; Mohammed, Ahmed S. A.; Caria, Joana; Sauter, Paul F.; Svergun, Dmitri I.; Lemke, Edward A.
• 署名单位: Johannes Gutenberg University of Mainz; Institute of Molecular Biology (IMB); European Molecular Biology Laboratory (EMBL); European Molecular Biology Laboratory (EMBL); Institute of Molecular Biology (IMB); Egyptian Knowledge Bank (EKB); Fayoum University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10403
• DOI: 10.1073/pnas.2415220121
• 发表日期: 2024-12-10
• 关键词: x-ray-scattering; small-angle scattering; single-molecule; conformational heterogeneity; phase-separation; fret; beamline; INFORMATION; acquisition; absorption

摘要

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 (RG), measured by small- angle X- ray scattering (SAXS), and the root mean square (rms) end- to- end distance (RE), 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 bling simultaneous and direct measurements of RG and RE 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 RE and RG varies under different denaturing conditions, highlighting discrepancy rather than shortcomings in either of the two established methods. The 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.