Site-specific dynamic nuclear polarization of hydration water as a generally applicable approach to monitor protein aggregation

Abstract

We present a generally applicable approach for monitoring protein aggregation by detecting changes in surface hydration water dynamics and the changes in solvent accessibility of specific protein sites, as protein aggregation proceeds in solution state. This is made possible through the Overhauser dynamic nuclear polarization (DNP) of water interacting with stable nitroxide spin labels tethered to specific proteins sites. This effect is highly localized due to the magnetic dipolar nature of the electron-proton spin interaction, with >80% of their interaction occurring within 5 A between the unpaired electron of the spin label and the proton of water. We showcase our tool on the aggregation of tau proteins, whose fibrillization is linked to neurodegenerative disease pathologies known as taupathies. We demonstrate that the DNP approach to monitor local changes in hydration dynamics with residue specificity and local contrast can distinguish specific and neat protein-protein packing leading to fibers from non-specific protein agglomeration or precipitation. The ability to monitor tau assembly with local, residue-specific, resolution, under ambient conditions and in solution state will help unravel the mechanism and structural characteristics of the gradual process of tau aggregation into amyloid fibers, which remains unclear to this day.

Fig 1

A. Diagramatic representation of tau¹⁸⁷: the N-terminally truncated tau fragment that contains residues 255–441 of the longest human tau isoform and includes all four MTB repeat domains R1–R4. B. Time dependence of ¹H DNP-enhanced signal of hydration water as probed through MTSL spin-labeled cites C322 and S413C of tau¹⁸⁷. C. Transmission electron micrographs showing the different morphologies of the aggregates formed by tau¹⁸⁷C322-SL and tau¹⁸⁷S413C-SL after 16–24 hours of heparin-induced aggregation.

Fig. 2

Time dependence of ¹H DNP signal intensity and EPR signal intensity of the center (I¹⁴N=1) hyperfine line probed at site C322-SL, during 10 hours of aggregation of tau¹⁸⁷ with application of the same microwave power of 128 mW for DNP saturation and detection of EPR resonances. This intermediate power setting provides insufficient power for DNP, but too much power to obtain an optimally narrow EPR signal.

Fig. 3

Time dependence of ¹H DNP signal intensity and EPR signal intensity of the center (I¹⁴N=1) hyperfine line probed at site C322-SL, during 10 hours of aggregation of tau¹⁸⁷ with application of optimal (close to extrapolated maximum) power setting for ¹H DNP and optimal (non-saturating) power setting for EPR/