Assigning Backbone NMR Resonances for Full Length Tau Isoforms: Efficient Compromise between Manual Assignments and Reduced Dimensionality

Tau protein is the longest disordered protein for which nearly complete backbone NMR resonance assignments have been reported. Full-length tau protein was initially assigned using a laborious combination of bootstrapping assignments from shorter tau fragments and conventional triple resonance NMR experiments. Subsequently it was reported that assignments of comparable quality could be obtained in a fully automated fashion from data obtained using reduced dimensionality NMR (RDNMR) experiments employing a large number of indirect dimensions. Although the latter strategy offers many advantages, it presents some difficulties if manual intervention, confirmation, or correction of the assignments is desirable, as may often be the case for long disordered and degenerate polypeptide sequences. Here we demonstrate that nearly complete backbone resonance assignments for full-length tau isoforms can be obtained without resorting either to bootstrapping from smaller fragments or to very high dimensionality experiments and automation. Instead, a set of RDNMR triple resonance experiments of modest dimensionality lend themselves readily to efficient and unambiguous manual assignments. An analysis of the backbone chemical shifts obtained in this fashion indicates several regions in full length tau with a notable propensity for helical or strand-like structure that are in good agreement with previous observations

¹³C/¹H strip plots at ¹⁵N chemical shifts of 119.06 ppm (A), 116.36 ppm (B), and 123.59 ppm (C) extracted from the (4,3)D HNCACB experiment for Tau441 (only the additive strips are shown) corresponding to residues His94, Thr95, and Glu96.

<p>Dashed lines indicate the inter-residue connectivities. Assignments were verified using the additive (D) and subtractive (E) linear combinations of the (4,3)D HNNCANNH data as shown by ¹⁵N/¹H strip plots below at the ¹⁵N chemical shift of Thr95 at 116.58.</p

¹³C/¹H strip plots from 3D (left) and (4,3)D (right; additive experiment) HNCACB experiments, at ¹⁵N chemical shifts of 125.69 ppm and 125.67 ppm, respectively, for Tau K19 K257T.

<p>Shown are the spin systems for residues Gln307 and Glu338 (corresponding to tau352 residues 218 and 249) which can be better and more accurately resolved and identified using the GFT linear combinations. Positive peaks are shown in black and negative peaks are in red.</p

Diagram of the full-length tau protein, Tau441 showing the domain structure, alternately spliced exons (green) and the microtubule-associated repeats (R1–R4).

<p>Tau352 is the shortest full-length form of tau with no alternately spliced regions. TauK19 containing R1, R3 and R4 is shown above.</p

Acquisition Parameters of GFT-NMR Experiments.

†<p>Data collected at 600 MHz.</p>§<p>Data collected at 900 MHz.</p>‡<p>Experimental details published in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034679#pone.0034679-Atreya1" target="_blank">[35]</a>.</p>*<p>For this experiment Ω(C^α) was detected in quadrature.</p

¹⁵N/¹H HSQC spectrum for free state tau352 with peak assignments given by residue numbers for the longest tau isoforms (tau441).

<p>The highly overlapped region (box) is enlarged.</p

¹³C/¹H strip plots showing inter-residue connectivities for residues Val128, Ser129, and Lys130 from the (4,3)D HNCACB additive (A) and subtractive (B) data for Tau352 at, at ¹⁵N chemical shifts of (1) 122.31 ppm (2) 120.14 ppm and (3) 124.10 ppm.

<p>Assignments were made by walking through the data from <i>i</i> to <i>i−1</i> spin systems. Positive peaks are shown in black and negative peaks are in red. Dashed lines show peak connectivities.</p

Correlation plot of the C^α chemical shift values from the (4,3)D HNCACB experiment versus those from the standard 3D HNCACB for tauK19 K257T.

<p>The lease squares fit has a slope of 1.001 and an R² value of 0.999.</p

CSI plots for Tau352 (A) full sequence and (B) microtubule binding region (bars) overlaid with data for TauK19 (red line).

<p>Positive deviations are indicative of α-helical conformations while negative deviations correspond to β-sheet structure.</p