Automatic assignment of the intrinsically disordered protein Tau with 441-residues

Intrinsically disordered proteins carry out many important functions in the cell. However, the lack of an ordered structure causes dramatic signal overlap and complicates the NMR-based characterization of their structure and dynamics. Here we demonstrate that the resonance assignment of 441-residue Tau and its smaller isoforms, htau24 (383 residues) and htau23 (352 residues), three prototypes of intrinsically disordered proteins, which bind to microtubules and play a key role in Alzheimer disease, can be obtained within 5 days by a combination of seven-dimensional NMR spectra with optimized methods for automatic assignment. Chemical shift differences between the three isoforms provide evidence for the global folding of Tau in solution

Recombinant ncTom40 has a β-barrel structure. (a) Far UV CD spectra of ncTom40 in decylmaltoside. (b) Superposition of ¹³C-¹³C proton driven spin diffusion spectra of ncTom40 (red) and hVDAC1 (green; reproduced from [4]), both in DMPC liposomes. The mixing time was 15 ms.

<p>Recombinant ncTom40 has a β-barrel structure. (a) Far UV CD spectra of ncTom40 in decylmaltoside. (b) Superposition of ¹³C-¹³C proton driven spin diffusion spectra of ncTom40 (red) and hVDAC1 (green; reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112374#pone.0112374-Schneider1" target="_blank">[4]</a>), both in DMPC liposomes. The mixing time was 15 ms.</p

Structural characterization of liposome-embedded ncTom40 by H/D exchange coupled to solution-state NMR.

<p>(a) Enlarged spectral regions of [¹H, ¹⁵N]-HSQC spectra at increasing back-exchange times. To reduce signal overlap, ncTom40 was selectively ¹⁵N-labeled at ALA, HIS, ILE, MET, THR. Time points indicate the time after start of the first HSQC. The dissolution buffer contained 75% D₂O. Sequence-specific resonance assignments are indicated. (b) Sequence-specific signal intensities in the first HSQC after dissolution in 100% D₂O buffer. (c) NMR signal intensity change of residues in panel (a) during back-exchange in 75% D₂O. Intensity values were normalized on the basis of the noise level in the spectra. Error bars are based on signal-to-noise. (d) Protonation ratios for residues of Tom40 at the beginning of back exchange. (e) Protonation ratios shown in (d) were mapped onto the topology model of ncTom40, which was predicted on the basis of its homology to hVDAC1. Residues predicted to be in a β-strand or α-helix are boxed. Green-shaded (red-shaded) residues have protonation ratios larger (lower) than 0.3. Residues shown in white were not analyzed due to signal overlap, low signal-to-noise or missing resonance assignment.</p

APSY experiments recorded at different temperatures and assignments obtained for denatured ncTom40 (339 non-proline residues) by MARS [31].

<p>Assignments classified by MARS as low are not reliable and were excluded from further analysis.</p><p>APSY experiments recorded at different temperatures and assignments obtained for denatured ncTom40 (339 non-proline residues) by MARS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112374#pone.0112374-Panchal1" target="_blank">[31]</a>.</p

Scheme illustrating the H/D exchange strategy developed for membrane proteins (blue) reconstituted into liposomes (yellow).

<p>A white color indicates H₂O buffer, black color 100% D₂O buffer and grey color the dissolution buffer, which contains 4 M GdnSCN. During the incubation period in 100% D₂O solvent exposed residues will exchange amide protons against deuterium (lower row, middle panel). They will therefore not be visible in the denatured monomer (lower right panel).</p

Cα secondary chemical shifts (upper chart) and Cα secondary structure propensities (lower chart) of ncTom40 obtained from APSY experiments recorded at 295K.

<p>Secondary structure propensities were calculated using SSP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112374#pone.0112374-Molday1" target="_blank">[42]</a>. The predicted topology of ncTom40 is shown on top with secondary structure elements highlighted in grey. Only assignments classified by MARS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112374#pone.0112374-Hiller1" target="_blank">[27]</a> as reliable were used.</p

NMR-Based Detection of Hydrogen/Deuterium Exchange in Liposome-Embedded Membrane Proteins.

Membrane proteins play key roles in biology. Determination of their structure in a membrane environment, however, is highly challenging. To address this challenge, we developed an approach that couples hydrogen/deuterium exchange of membrane proteins to rapid unfolding and detection by solution-state NMR spectroscopy. We show that the method allows analysis of the solvent protection of single residues in liposome-embedded proteins such as the 349-residue Tom40, the major protein translocation pore in the outer mitochondrial membrane, which has resisted structural analysis for many years.peerReviewe