Detection of Mutation-Induced Conformational Changes in an Intrinsically Disordered Protein by Double Quantum Coherence Electron Spin Resonance Methodology

Intrinsically disordered proteins (IDPs) underlie essential cellular functions and drive neurodegenerative diseases through mutation-induced structural changes, yet their conformational heterogeneity often evades crystallography and cryo-EM. Electron spin resonance (ESR) pulsed dipolar spectroscopy (PDS), which determines distance distributions between a pair of spin-labeled residues in a protein, can provide complementary and meaningful information related to conformational heterogeneity in IDPs. Double quantum coherence (DQC) is an important ESR PDS technique, capable of measuring a wide range of distances (∼10 to at least 80 Å), and is a single-frequency technique with a small background that can be easily removed. This makes DQC an ideal candidate to probe IDPs. We present a complete theoretical framework for DQC data analysis, incorporating pseudosecular dipolar coupling and finite pulse effects, enabling rapid and accurate reconstruction of complex distance distributions in doubly nitroxide-labeled IDPs. We validate the method on rigid biradicals with known interspin distances. The application to a tau protein fragment (jR2R3) reveals distinct end-to-end distance distributions for the wild-type vs the disease-associated P301L mutant. The results expose differences in their conformational distributions, which likely govern their divergent aggregation propensities. This advance also establishes DQC ESR as a powerful, accessible tool for probing disorders in biomolecular systems.