Journal: International Journal of Molecular Sciences

Article Title: Impact of the Hereditary P301L Mutation on the Correlated Conformational Dynamics of Human Tau Protein Revealed by the Paramagnetic Relaxation Enhancement NMR Experiments

doi: 10.3390/ijms21113920

Figure Lengend Snippet: ( A ) The domain architecture of the longest isoform of the human tau protein, which comprises 441 residues (hTau441), and the domain architecture of the fragment (TauF4Δ) used in this work. N1 and N2 are acidic regions (cyan), P1 and P2 are proline-rich regions (yellow). R1, R2, R3 and R4 are the pseudo-repeats (purple) that engage in microtubule binding and constitute the core of tau filament structures. The two hexapeptides highlighted in green are essential for tau fibrillization, which are named PHF6* and PHF6. ( B ) The primary sequence of TauF4Δ. The secondary structures found in the heparin-induced tau filament structure are indicated on the primary sequence and the dotted line indicates the unstructured part in the tau filament . Two PGGG sequences near the amyloid motifs (PHF6* and PHF6 in green) are highlighted in gray, in which P301 is marked in red. The residues in yellow are the positions used for MTSL spin-labeling.

Article Snippet: The gene fragment coding TauF4Δ (residues 225–324) was cloned into the expression vector pET28a (Addgene, Watertown, MA, USA).

Techniques: Binding Assay, Sequencing, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Impact of the Hereditary P301L Mutation on the Correlated Conformational Dynamics of Human Tau Protein Revealed by the Paramagnetic Relaxation Enhancement NMR Experiments

doi: 10.3390/ijms21113920

Figure Lengend Snippet: Comparing the conformational dynamic parameters between the wild-type and P301L mutant TauF4Δ. ( A ) Heteronuclear 1 H- 15 N nuclear Overhauser effects (hNOEs) are plotted. The upper and lower panels are the wild-type and P301L mutant TauF4Δ.The domain architecture of tau and the secondary structures formed in the heparin-induced tau filament are drawn on each plot. hNOE values that are larger than the average value (blue line) are marked in red. The gray bars indicate the positions for β3 and β5. ( B ) Residues with hNOE values greater than the average are marked by red circles on the heparin-induced tau filament structure (PDB ID: 6QJH) . ( C ) R 2 / R 1 values; the wild-type (upper) and the P301L mutant (lower). R 2 / R 1 values shown in red are higher than the average value (blue line). Gray bars mark the position for the linker connecting β2 and β3. ( D ) Residues with R 2 / R 1 values above the average are marked by red circles on the tau filament structure.

Article Snippet: The gene fragment coding TauF4Δ (residues 225–324) was cloned into the expression vector pET28a (Addgene, Watertown, MA, USA).

Techniques: Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Impact of the Hereditary P301L Mutation on the Correlated Conformational Dynamics of Human Tau Protein Revealed by the Paramagnetic Relaxation Enhancement NMR Experiments

doi: 10.3390/ijms21113920

Figure Lengend Snippet: Secondary chemical shifts of TauF4Δ. ( A ) Secondary chemical shifts for 13 C α , ΔδC α and the averaged secondary chemical shifts for 13 C α and 13 C’, Δδ av (C α C’), for the wild-type are shown in the left panels. The expanded plots around the mutation site, P301, are placed in the right panels. The averaged secondary chemical shits are calculated as [3ΔδC α + 4ΔδC’]/7 . Regions showing β-structure propensity are identified by negative Δδ av (C α C’) values that extend over several residues and are marked in red. ( B ) The secondary chemical shifts ΔδC α and Δδ av (C α C’) for the P301L mutant are presented. The gray bar represents the residue did not show a resolved signal.

Article Snippet: The gene fragment coding TauF4Δ (residues 225–324) was cloned into the expression vector pET28a (Addgene, Watertown, MA, USA).

Techniques: Mutagenesis, Residue

Journal: International Journal of Molecular Sciences

Article Title: Impact of the Hereditary P301L Mutation on the Correlated Conformational Dynamics of Human Tau Protein Revealed by the Paramagnetic Relaxation Enhancement NMR Experiments

doi: 10.3390/ijms21113920

Figure Lengend Snippet: PRE correlation maps for the ( A ) wild-type and ( B ) P301L mutant of TauF4Δ. Colors indicate correlated (red), anti-correlated (blue) and uncorrelated (green) contours. Only residue positions with observable PRE rates for two or more mutants are given. MTSL labeling positions are shown on the maps by the red circles filled in dark blue. The segments with locally correlated residues are marked by the blue squares labeled C1–C8.

Article Snippet: The gene fragment coding TauF4Δ (residues 225–324) was cloned into the expression vector pET28a (Addgene, Watertown, MA, USA).

Techniques: Mutagenesis, Residue, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Impact of the Hereditary P301L Mutation on the Correlated Conformational Dynamics of Human Tau Protein Revealed by the Paramagnetic Relaxation Enhancement NMR Experiments

doi: 10.3390/ijms21113920

Figure Lengend Snippet: PRI correlation maps for the ( A ) wild-type and ( B ) P301L mutant of TauF4Δ. Only residues with observable PRI rates for two variants are shown. The red circles filled in dark blue indicate the positions of the MTSL labels. The MTSL positions connected by orange dotted lines are used for simultaneous spin-labeling. Segments comprising residues with local correlations are marked by the blue squares labeled by the closely corresponding segments in the PRE map. Colors of the contours indicate the degrees of correlations as used in the PRE maps: correlated (red), anti-correlated (blue) and uncorrelated (green).

Article Snippet: The gene fragment coding TauF4Δ (residues 225–324) was cloned into the expression vector pET28a (Addgene, Watertown, MA, USA).

Techniques: Mutagenesis, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Impact of the Hereditary P301L Mutation on the Correlated Conformational Dynamics of Human Tau Protein Revealed by the Paramagnetic Relaxation Enhancement NMR Experiments

doi: 10.3390/ijms21113920

Figure Lengend Snippet: Comparison of PRI rates (Δ 1 H N -Γ 2 ) between the wild-type and P301L mutant TauF4Δ. ( A ) PRI rates for the wild-type (black bars) and P301L mutant (red bars) TauF4Δ labeled at residues 262 and 305. The black arrows indicate the positions of the MTSL labels. ( B ) The correlations of the PRI rates between the wild-type and P31L mutant labeled at 262 and 305 are mapped. Residues showing significant differences in rates between the two proteins are labeled. Labeling in red shows that the PRI rates have increased in the P301L mutant when compared with that of the wild-type, whereas labeling in blue indicates that the PRI rate decreased in the mutant when compared with that of the wild-type protein. ( C ) Residues having significantly changed PRI rates are indicated by the red circles on the heparin-induced tau filament structure (PDB ID: 6QJH) . The colors of the residue numbers represent the same meanings as used in the PRI rate correlations. The yellow circles indicate residues that are MTSL labeled. ( D ) The PRI rates for the wild-type and P301L mutant TauF4Δ labeled at residues 291 and 322. ( E ) The PRI rate correlations between the wild-type and the P301L mutant labeled at 291 and 322. ( F ) Residues with significant changes in PRI rates in the P301L mutant are shown. Gray bars represent the residues that did not show the resolved signals on the spectra.

Article Snippet: The gene fragment coding TauF4Δ (residues 225–324) was cloned into the expression vector pET28a (Addgene, Watertown, MA, USA).

Techniques: Comparison, Mutagenesis, Labeling, Residue

Journal: International Journal of Molecular Sciences

Article Title: Impact of the Hereditary P301L Mutation on the Correlated Conformational Dynamics of Human Tau Protein Revealed by the Paramagnetic Relaxation Enhancement NMR Experiments

doi: 10.3390/ijms21113920

Figure Lengend Snippet: Model structures of TauF4Δ based on significant changes in the PRI rates between the wild-type and P301L mutant. Three and two representative residues are selected from TauF4Δ labeled at 262 and 305 and TauF4Δ labeled at 291 and 322, respectively, all of which showed significant changes in PRI rates for the P301L mutant. The structures are modeled from the heparin-induced tau filament structure (PDB ID: 6QJH) to fulfill the presumed angle changes ( θ 1 – θ 5 ) suggested by the changes in the PRI rates. ( A ) A representative form of the wild-type TauF4Δ modeled based on the PRI rates for V275, N296 and K311 in the tau fragment with labeling at 262 and 305, and ( B ) also on the PRI rates for G271 and T319 in the tau fragment with labeling at 291 and 322. ( C ) A representative conformation similarly modeled based on the same set of data collected from the tau fragment labeled at residues 262 and 305, and ( D ) also for the tau fragment with labeling at 291 and 322 ( D ).

Article Snippet: The gene fragment coding TauF4Δ (residues 225–324) was cloned into the expression vector pET28a (Addgene, Watertown, MA, USA).

Techniques: Mutagenesis, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Impact of the Hereditary P301L Mutation on the Correlated Conformational Dynamics of Human Tau Protein Revealed by the Paramagnetic Relaxation Enhancement NMR Experiments

doi: 10.3390/ijms21113920

Figure Lengend Snippet: TauF4Δ segmental conformation dynamics determine the ensemble structures. The boxes represent segments that comprise residues that are undergoing correlated motions, which were identified on the PRE maps . Each label of the box indicates the corresponding segment name on the PRE maps. Residues in the segments C4–C8 closely overlap with residues in the β-structures (β1–β5). Segments corresponding to the amyloid motifs, PHF6* and PHF6, are green and other segments are purple. The conformations in the major ensembles of the wild-type and P301L mutant TauF4Δ should resemble each other, as indicated by the similar PRE maps . The P301L mutation destabilizes the β-turn structure of 301 PGGG 304 causing this region to adopt an extended structure preferentially. The conformational dynamics of the hinge linking C5 and C6 segments may further promote the extended form populations of the P301L mutant; the dynamic nature of the hinge was shown by the higher values of R 2 / R 1 ( C). The PRI-derived model structure of the wild-type TauF4Δ has moderately exposed PHF6, while the model for the P301L mutant largely exposes the amyloid motif, PHF6. The conformational ensemble switch explains the enhanced aggregation propensity of the P301L mutant. The disrupted inter-segment dynamics by the P301L mutation may facilitate conformational ensembles that differ from those of the wild-type.

Article Snippet: The gene fragment coding TauF4Δ (residues 225–324) was cloned into the expression vector pET28a (Addgene, Watertown, MA, USA).

Techniques: Mutagenesis, Derivative Assay