Quantum-dot-based single-molecule Förster resonance energy transfer for investigating the conformations of tau and phosphorylated tau

Quantum dots (QDs), with their tunable emission, high quantum yield, and superior photostability, have been widely employed as FRET donors or acceptors in ensemble measurements for transducting biomolecular interactions into measurable fluorescence signals. However, their implementation in single-molecule Förster resonance energy transfer (smFRET) remains limited, particularly for resolving intramolecular conformational dynamics. This gap stems from two inherent constraints: (1) their substantial hydrodynamic radii (5–15 nm) causing steric interference with biomolecular structures, and (2) stochastic bioconjugation approaches produce uncontrolled probe orientations that introduce uncertainty in FRET-derived distance calculations. Fluorescence cross-correlation spectroscopy (FCCS) overcomes these limitations by enabling solution-phase smFRET measurements, which provides an optimal framework for exploiting QDs in smFRET. Here, we challenged the QD-based smFRET system into measuring the conformational changes of intrinsically disordered tau proteins. Tau proteins were expressed with an N-terminal His-tag, enabling their direct and controllable immobilization on zinc-coated QDs via metal-coordination chemistry. The C-terminal Cys432 of mutant tau proteins were labeled with Cy5-maleimide as the acceptor to form a dual-labeled FRET system. Leveraging the large Förster distance (R 0  = 8.0 nm) of the QD605-Cy5 pair, pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) quantified the distance from the QD center to C432—a structural metric reporting on global compaction. Strikingly, phosphorylated tau (P-tau) reduced this distance from 10.0 ± 1.0 nm to 6.9 ± 1.3 nm, indicating obvious monomeric compaction. Our study establishes QDs as potent tools for smFRET studies of biomolecular conformations and provides a straightforward approach for investigating the single-molecule conformations of intrinsically disordered proteins in physical environment.