Rabdano, S.; Podkorytov, I.; Luzik, D.; Skrynnikov, N. Characterizing the Size of Protein Aggregate Particles Using a Combination of NMR Diffusion Measurements and Dynamic Light Scattering: Case Study of RRM2 Domain from Protein TDP-43. The FEBS Journal 2017, 284 (Suppl. 1), 212.
DOI: 10.1111/febs.14174.
The second RNA recognition motif (RRM2) from neuropathological protein TDP-43 is a small globular domain. Pulsed-field-gradient stimulated-echo (PFGSTE) NMR diffusion experiments and dynamic light scattering (DLS) measurements performed on a sample of RRM2 yield diffusion coefficients 1.38±0.01·10-10 and 1.40±0.08·10-10 m2/s respectively, in excellent agreement with theoretical predictions. In vitro oxidation of RRM2 leads to formation of disulfide-bonded dimers followed by domain unfolding and assembly of the protein into soluble aggregate particles (APs). These particles prove to be unstructured and highly inhomogeneous, preventing their direct observation by HSQC spectroscopy – except for RRM2 C-terminal tail which remains flexible and solvated. In particular, the C-terminal residue N76 produces a sharp signal with the same intensity as in the control (unoxidized) sample. This signal has been used for PFGSTE NMR measurements on the oxidized sample, yielding the diffusion coefficient 0.55±0.02·10‑10 m2/s. This is significantly different from the value determined by the DLS method, 0.40±0.01·10‑10 m2/s. The difference can be understood once we recognize that DLS and PFGSTE NMR experiments provide different sampling of the polydisperse AP ensemble. Specifically, DLS is more biased toward bigger particles (weighted according to r6, where r is the AP’s radius) compared to PFGSTE NMR (weighted according to r3 in the specific case when the signal originates from the fully flexible tail). Thus, the diffusion data from DLS and PFGSTE NMR measurements can be combined to characterize the size distribution function of the aggregate particles. We have shown that these data are consistent with the exponentially distributed r values and predict amply hydrated aggregate particles, ca. 3 grams of hydration water per gram of protein.
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