Test Your Hypothesis

What the evidence supports — and what it does not

The hypothesis conflates three separately demonstrated layers. First, ATP and hydrotrope analogs do shift monomer conformation in vitro: simulations and peptide assays show ATP increases monomer radius of gyration and suppresses β-sheet propensity in Aβ40, inhibiting dimerization and dissolving nucleating assemblies — but with supra-physiological ATP in simulations and peptide-centric readouts ATP monomer conformational shifts (MD + peptide assays). Second, hydrotrope effects on FET proteins are well quantified in vitro but only at the cluster level (DLS/NTA), not at the single-molecule level: FUS-SNAP hydrodynamic diameter rises non-monotonically with ATP•Mg²⁺ (281 → 441 → 1021 nm at 0, 0.5, 1 mM), and the authors themselves warn the SNAP-tag, buffer, and indirect size proxies limit in vivo extrapolation FET cluster data, in vitro only. Third, live-cell detection of TDP-43 monomerization exists — but via a bimolecular luminescence complementation reporter (TDP-DiLuc), not smFRET, and monomerization is a dimerization-state readout, not a folded-domain conformational shift TDP-DiLuc live-cell monomerization reporter.

The missing data points above are intentional: the source reports higher ATP concentrations qualitatively, and inventing values would misrepresent the evidence.

Battle-testing the smFRET-in-neurons premise

Critical unknowns, per BGPT inference from the cited records: (1) intraneuronal protein concentrations are incompatible with sparse single-molecule imaging unless endogenous expression is knocked down and a low-copy knock-in is used — the CRISPR endogenous-tagging route achieved only ~4.7% mAvicFP1-positive HEK293T cells and needed FACS cloning Endogenous tagging feasibility in cells; (2) hydrotrope analogs may perturb dye photophysics and protein-dye linkages, confounding FRET efficiency changes; (3) hydrotrope effects are non-monotonic, so a single dose cannot be interpreted thermodynamically; (4) no record demonstrates smFRET inside any live neuron. The strongest counter-hypothesis: any FRET shift observed would reflect altered oligomerization/cluster state (as in the FET data) rather than a monomer conformational change — the FET study explicitly attributes effects to non-specific amphiphile-protein interactions, not hydrotropy per se.

What would falsify or confirm it

Confirm: purify a dual-cysteine-labeled FUS or TDP-43 construct, show ATP•Mg²⁺-dependent FRET efficiency shifts in vitro matching the conformational-expansion prediction, then reproduce the shift in knock-in neurons with LC-MS-verified intracellular analog levels and PQC-inhibited controls. Falsify: FRET changes disappear when cluster formation is blocked (mutating crosslinking interfaces) — implying the signal is oligomeric, not monomeric. Confidence: moderate; the conformational-shift premise is in vitro-supported, but the live-neuron smFRET step is entirely untested and the nearest live-cell analog (TDP-DiLuc) measures dimerization, not folding.