NMR spectroscopy of Tau, a naturally unfolded protein

When we first saw a spectrum of Tau we believed that the NMR of Tau was hopeless (look at the spectrum !). The signal dispersion is very poor, with the protons signals occupying only 1 ppm. Still, we managed to get sequence specific assignment of 40% of the residues, and have since been looking at every aspects of its biological function (microtubule binding) and misfunction (aggregation) by NMR. NMR spectroscopy of the full-length neuronal Tau protein has proved difficult due to the length of the protein and the unfavorable amino acid composition. We have shown that the random-coil chemical shift values and their dependence on the presence of a proline residue in the (i+1) position can successfully be exploited to assign all proline-directed phosphorylation sites. This is a first step toward the study of the phosphorylation of Tau by NMR spectroscopy. A combined strategy to obtain a partial NMR assignment of the neuronal Tau protein was presented. Confronted with the extreme spectral degeneracy that the spectrum of this 441 amino acid long unstructured protein presents, we have introduced a graphical procedure based on residue type-specific product planes. Combining this strategy with the search for pairwise motifs, and combining the spectra of different Tau isoforms and even of peptides derived from the native sequence, we arrive at a partial assignment that is sufficient to map the interactions of Tau with its molecular partners.


The Tau protein - 1H-15N HSQC spectrum

In vivo, Tau in PHF is found in its hyperphosphorylated form. In vitro, the addition of poly-anions (heparin, arachidonic acid, RNA) leads to aggregation at 37°C. We used the HRMAS technology to observe the fibers. We had to choose low spinning rates such as not to destroy the fibers. Electron microsocopy was used to verify the structural integrity after the experiment.

Electron microscopy of the PHFs before spinning (left), after prolonged spinning at 1400 Hz (middle) and 6 kHz (right).
In vitro aggregated Tau with heparin (16kD, Sigma) at 37°C, 300 µDTT (in black) HRMAS NMR in 4mm rotor at 600 or 800MHz field strength.


NMR of Tau mature fibers

For Tau integrated into the fibers Intensity differences relate to differential mobility and allow to define the core region of Tau integrated into the fibers.

In vitro aggregated Tau with heparin (16kD, Sigma) at 37°C, 300 µDTT liquid state NMR with cryoprobe at 600MHz
Tau_6 Tau_6
Schematic view of Tau assembled into mature PHFs. The frequency of the sine wave indicates the residual mobility, where this term covers both rapid and slow motions. The first highly mobile zone extends from the N-terminal to ala77. A second zone of linearly reduced mobility covers the primary sequence up to Gly261. The rigid core, defined by a residual intensity inferior to 10%, extends to Thr377. Finally, a second zone of reduced mobility covers the C-terminal part of Tau.

How does heparin promote PHF formation?

We use NMR to characterize the interaction between heparin and Tau, first in a qualitative way: here you can see overlaid HSQC spectrum corresponding to addition of heparin on Tau.The resonances of the lysines involved in the interaction are shifted. This interaction can be quantified by calculating a Kd based on the observed gradual shift of resonances during the titration. When forming the fibers by parallel in-register stacking, an inter-molecular poly-lysine peptide forms. The poly-anion is required to charge-compensate for this. Charge neutralization as a result of binding to the two regions up- and downstream of the Microtubule Binding Region, which were proposed to inhibit the aggregation process, well mimicks the hyperphosphorylation that characterizes in vivo generated PHF-Tau, but we still have to confirm in vitro how phosphorylation does induces the aggregation.

In vitro interaction between 15N-Lys, 13C Tau with heparin (4kD, gift from B. Mulloy) at 20°C, 300 µDTT Titration by liquid state NMR with cryoprobe at 600MHz.

Use NMR spectroscopy to characterize in vitro phosphorylated Tau samples using recombinant kinases.

There are a lot of phosphorylation sites on the Tau protein and some of these sites are specifically phosphorylated in diseased neurons in Alzheimer's brains. The phosphorylation of Tau is however also a normal process that will regulate the binding of Tau to the microtubules. The phosphorylation decreases the capacity of Tau to polymerize the microtubules. We can map the phosphorylation sites by NMR, for example here by using the PKA kinase, and use the well characterized phosphorylated sample for functional assays like agregation (fluorescence of ThioS) or tubuline polymerization (turbidity) and interaction with other proteins (NMR chemical shift perturbation).

Phosphorylation of Tau by PKA on Ser214