Tau In Solution Research Articles

Insight into the Structure of the “Unstructured” Tau Protein

Combining structural proteomics experimental data with computational methods is a powerful tool for protein structure prediction. Here, we apply a recently developed approach for de novo protein structure determination based on the incorporation of short-distance crosslinking data as constraints in discrete molecular dynamics simulations (CL-DMD), for the determination of the conformational ensemble of tau protein in solution. The predicted structures were inagreement with surface modification and long-distance crosslinking data. Tau in solution was found as an ensemble of rather compact globular conformations with distinct topology, inter-residue contacts, and a number of transient secondary-structure elements. Regions important for pathological aggregation consistently were found to contain β strands. The determined structures are compatible with the tau protein in solution being a molten globule at near-ground state with persistent residual structural features which we were able to capture by CL-DMD. The predicted structure may facilitate an understanding of the misfolding and oligomerization pathways of the tau protein.

Global Conformation of Tau Protein Mapped by Raman Spectroscopy.

Alzheimer's disease (AD) is one of the neurodegenerative disease characterized by progressive neuronal loss in the brain. Its two major hallmarks are extracellular senile plaques and intracellular neurofibrillary tangles (NFTs), formed by aggregation of amyloid β-42 (Aβ-42) and Tau protein respectively. Aβ-42 is a transmembrane protein, which is produced after the sequential action of β- and γ-secretases, thus obtained peptide is released extracellularly and gets deposited on the neuron forming senile plaques. NFTs are composed of microtubule-associated protein-Tau (MAPT). Tau protein's major function is to stabilize the microtubule that provides a track on which the cargo proteins are shuttled and the stabilized microtubule also maintains shape and integrity of the neuronal cell. Tau protein is subjected to various modifications such as phosphorylation, ubiquitination, glycation, acetylation, truncation, glycosylation, deamination, and oxidation; these modifications ultimately lead to its aggregation. Phosphorylation is the major modification and is extensively studied with respect to Tau protein. Tau protein, however, undergoes certain level of phosphorylation and dephosphorylation, which regulates its affinity for microtubule and ultimately leading to microtubule assembly and disassembly. Our main aim was to study the native state of longest isoform of Tau (hTau40WT-4R2N) and its shortest isoform, (hTau23WT-3R0N), at various temperatures such as 10, 25, and 37 °C. Raman spectroscopic results suggested that the proportion of random coils or unordered structure depends on the temperature of the protein environment. Upon increase in the temperature from 10 to 37 °C, the proportion of random coils or unordered structures increased in the case of hTau40WT. However, we did not find a significant effect of temperature on the structure of hTau23WT. This current approach enables one to analyze the global conformation of soluble Tau in solution.

Intrinsically Disordered Proteins in Neurodegeneration

Intrinsically disordered proteins (IDPs) have gained wide recognition due to their versatile roles in cell physiology and pathology. A large repertoire of IDPs has been implicated in numerous diseases, making them potential targets for therapeutic intervention. An IDP, which plays a crucial role in a variety of neurodegenerative diseases, is the protein Tau. As a microtubule-associated protein, Tau regulates the assembly and dynamics of microtubules. In the brain of Alzheimer disease patients, Tau is hyperphosphorylated and insoluble deposits of Tau correlate with disease progression. Using NMR spectroscopy as the primary tool we have studied the dynamic ensemble of conformations of Tau in solution, the modulation of dynamic Tau structure by phosphorylation, the structure of Tau aggregates, the mechanism of Tau aggregation inhibitors as well as the interaction of Tau with microtubules and molecular chaperones. To this end we developed improved methods for NMR resonance assignment and ensemble determination. In this presentation I will present recent progress in our understanding of the structural polymorphism of Tau.

Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules

Microtubules (MTs) are hollow cytoskeletal filaments assembled from αβ-tubulin heterodimers. Tau, an unstructured protein found in neuronal axons, binds to MTs and regulates their dynamics. Aberrant Tau behavior is associated with neurodegenerative dementias, including Alzheimer's. Here, we report on a direct force measurement between paclitaxel-stabilized MTs coated with distinct Tau isoforms by synchrotron small-angle X-ray scattering (SAXS) of MT-Tau mixtures under osmotic pressure (P). In going from bare MTs to MTs with Tau coverage near the physiological submonolayer regime (Tau/tubulin-dimer molar ratio; ΦTau = 1/10), isoforms with longer N-terminal tails (NTTs) sterically stabilized MTs, preventing bundling up to PB ∼ 10,000-20,000 Pa, an order of magnitude larger than bare MTs. Tau with short NTTs showed little additional effect in suppressing the bundling pressure (PB ∼ 1,000-2,000 Pa) over the same range. Remarkably, the abrupt increase in PB observed for longer isoforms suggests a mushroom to brush transition occurring at 1/13 < ΦTau < 1/10, which corresponds to MT-bound Tau with NTTs that are considerably more extended than SAXS data for Tau in solution indicate. Modeling of Tau-mediated MT-MT interactions supports the hypothesis that longer NTTs transition to a polyelectrolyte brush at higher coverages. Higher pressures resulted in isoform-independent irreversible bundling because the polyampholytic nature of Tau leads to short-range attractions. These findings suggest an isoform-dependent biological role for regulation by Tau, with longer isoforms conferring MT steric stabilization against aggregation either with other biomacromolecules or into tight bundles, preventing loss of function in the crowded axon environment.

The Conformational Ensembles of α-Synuclein and Tau: Combining Single-Molecule FRET and Simulations

Intrinsically disordered proteins (IDPs) are increasingly recognized for their important roles in a range of biological contexts, both in normal physiological function and in a variety of devastating human diseases. However, their structural characterization by traditional biophysical methods, for the purposes of understanding their function and dysfunction, has proved challenging. Here, we investigate the model IDPs α-Synuclein (αS) and tau, that are involved in major neurodegenerative conditions including Parkinson’s and Alzheimer’s diseases, using excluded volume Monte Carlo simulations constrained by pairwise distance distributions from single-molecule fluorescence measurements. Using this, to our knowledge, novel approach we find that a relatively small number of intermolecular distance constraints are sufficient to accurately determine the dimensions and polymer conformational statistics of αS and tau in solution. Moreover, this method can detect local changes in αS and tau conformations that correlate with enhanced aggregation. Constrained Monte Carlo simulations produce ensembles that are in excellent agreement both with experimental measurements on αS and tau and with all-atom, explicit solvent molecular dynamics simulations of αS, with much lower configurational sampling requirements and computational expense.

Tau Protein Diffuses along the Microtubule Lattice

Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.

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.

Inhibition of Tau Polymerization with a Cyanine Dye in Two Distinct Model Systems

In a host of neurodegenerative diseases Tau, a microtubule-associated protein, aggregates into insoluble lesions within neurons. Previous studies have utilized cyanine dyes as Tau aggregation inhibitors in vitro. Herein we utilize cyanine dye 3,3'-diethyl-9-methyl-thiacarbocyanine iodide (C11) to modulate Tau polymerization in two model systems, an organotypic slice culture model derived from Tau transgenic mice and a split green fluorescent protein complementation assay in Tau-expressing cells. In slice cultures, submicromolar concentrations (0.001 microm) of C11 produced a significant reduction of aggregated Tau and a corresponding increase in unpolymerized Tau. In contrast, treatment with a 1 microm dose promoted aggregation of Tau. These results were recapitulated in the complementation assay where administration of 1 microm C11 produced a significant increase in polymerized Tau relative to control, whereas treatment of cells with 0.01 microm C11 resulted in a marked reduction of aggregated Tau. In the organotypic slice cultures, modulation of Tau aggregation was independent of changes in phosphorylation at disease and microtubule binding relevant epitopes for both dosing regimes. Furthermore, treatment with 0.001 microm C11 resulted in a decrease in both total filament mass and number. There was no evidence of apoptosis or loss of synaptic integrity at either dose, however, whereas submicromolar concentrations of C11 did not interfere with microtubule binding, higher doses resulted in a decrease in the levels of microtubule-bound Tau. Overall, a cyanine dye can dissociate aggregated Tau in an ex vivo model of tauopathy with little toxicity and exploration of the use of these type of dyes as therapeutic agents is warranted.

The Natively Unfolded Character of Tau and Its Aggregation to Alzheimer-like Paired Helical Filaments

The abnormal aggregation of the microtubule-associated protein Tau into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer disease (AD). Tau in solution behaves as a natively unfolded or intrinsically disordered protein while its aggregation is based on the partial structural transition from random coil to beta-structure. Our aim is to understand in more detail the unfolded nature of Tau, to investigate the aggregation of Tau under different conditions and the molecular interactions of Tau in filaments. We show that soluble Tau remains natively unfolded even when its net charge is minimized, in contrast to other unfolded proteins. The CD signature of the random-coil character of Tau shows no major change over wide variations in charge (pH), ionic strength, solvent polarity, and denaturation. Thus there is no indication of a hydrophobicity-driven collapse, neither in the microtubule-binding repeat domain constructs nor in full-length Tau. This argues that the lack of hydrophobic residues but not the net charge accounts for unfolded nature of soluble Tau. The aggregation of the Tau repeat domain (that forms the core of PHFs) in the presence of nucleating polyanionic cofactors (heparin) is efficient in a range of buffers and pH values between approximately 5 and 10 but breaks down beyond that range, presumably because the pattern of charged interactions disappears. Similarly, elevated ionic strength attenuates aggregation, and the temperature dependence is bell-shaped with an optimum around 50 degrees C. Reporter dyes ThS and ANS record the aggregation process but sense different states (cross-beta-structure vs hydrophobic pockets) with different kinetics. Preformed PHFs are surprisingly labile and can be disrupted by denaturants at rather low concentration ( approximately 1.0 M GdnHCl), much less than required to denature globular proteins. Partial disaggregation of Tau filaments at extreme pH values monitored by CD and EM indicate the importance of salt bridges in filament formation. In contrast, Tau filaments are remarkably resistant to high temperature and high ionic strength. Overall, the stability of PHFs appears to depend mainly on directed salt bridges with contributions from hydrophobic interactions as well, consistent with a recent structural model of the PHF core derived from solid state NMR (Andronesi, O. C., von Bergen, M., Biernat, J., Seidel, K., Griesinger, C., Mandelkow, E., and Baldus, M. (2008) Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy.

Domain Conformation of Tau Protein Studied by Solution Small-Angle X-ray Scattering

Tau is one of the two main proteins involved in the pathology of Alzheimer's disease via formation of beta-sheet rich intracellular aggregates named paired helical filaments (PHFs). Given that tau is a natively unfolded protein with no folded core (even upon binding to physiological partners such as microtubules), its structural analysis by high-resolution techniques has been difficult. In this study, employing solution small-angle X-ray scattering from the full length isoforms and from a variety of deletion and point mutants the conformation of tau in solution is structurally characterized. A recently developed ensemble optimization method was employed to generate pools of random models and to select ensembles of coexisting conformations, which fitted simultaneously the scattering data from the full length protein and deletion mutants. The analysis of the structural properties of these selected ensembles allowed us to extract information about residual structure in different domains of the native protein. The short deletion mutants containing the repeat domain (considered the core constituent of the PHFs) are significantly more extended than random coils, suggesting an extended conformation of the repeat domain. The longer tau constructs are comparable in size with the random coils, pointing to long-range contacts between the N- and C-termini compensating for the extension of the repeat domain. Moreover, most of the aggregation-promoting mutants did not show major differences in structure from their wild-type counterparts, indicating that their increased pathological effect is triggered only after an aggregation core has been formed.

FRET analysis of the conformation of tau in solution

FRET analysis of the conformation of tau in solution

Folding of the Repeat Domain of Tau Upon Binding to Lipid Surfaces

The microtubule-associated protein tau is impacted in neurodegeneration and dementia through its deposition in the form of paired helical filaments in Alzheimer's disease neurofibrillary tangles and through mutations linking it to the autosomal dominant disorder frontotemporal dementia with Parkinsonism. When isolated in solution tau is intrinsically unstructured and does not fold, while the conformation of the protein in the microtubule-bound state remains uncharacterized. Here we show that the repeat region of tau, which has been reported both to mediate tau microtubule interactions and to constitute the proteolysis-resistant core of disease-associated tau aggregates, associates with lipid micelles and vesicles and folds into an ordered structure upon doing so. In addition to providing the first structural insights into a folded state of tau, our results support a role for lipid membranes in mediating tau function and tau pathology.

P3-316: Fret analysis of the conformation of tau in solution

P3-316: Fret analysis of the conformation of tau in solution

Global Hairpin Folding of Tau in Solution

The microtubule-associated protein tau stabilizes microtubules in its physiological role, whereas it forms insoluble aggregates (paired helical filaments) in Alzheimer's disease. Soluble tau is considered a natively unfolded protein whose residual folding and intramolecular interactions are largely undetermined. In this study, we have applied fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) to examine the proximity and flexibility of tau domains and the global folding. FRET pairs spanning the tau molecule were created by inserting tryptophans (donor) and cysteines (labeled with IAEDANS as an acceptor) by site-directed mutagenesis. The observed FRET distances were significantly different from those expected for a random coil. Notably, the C-terminal end of tau folds over into the vicinity of the microtubule-binding repeat domain, the N-terminus remains outside the FRET distance of the repeat domain, yet both ends of the molecule approach one another. The interactions between the domains were obliterated by denaturation in GdnHCl. Paramagnetic spin-labels attached in various domains of tau were analyzed by EPR and exhibited a high mobility throughout. The data indicate that tau retains some global folding even in its "natively unfolded" state, combined with the high flexibility of the chain.

Structure, microtubule interactions, and phosphorylation of tau protein.

This paper summarizes recent structural and functional studies on tau protein, its interactions with microtubules, its self-assembly into paired helical filaments (PHF)-like fibers, and its modification by phosphorylation. The structure of tau in solution resembles that of a random coil. Both tau and Alzheimer PHFs have very little secondary structure, making it improbable that the assembly of tau into PHFs is based on interacting beta sheets. Tau's binding to microtubules can be described by a "jaws" effect. The domain containing the repeats binds very weakly, while the flanking regions (jaws) bind strongly, even without the repeats. However, only the combination of flanking regions and repeats makes binding productive in terms of microtubule nucleation and assembly. Although the majority of Alzheimer-like phosphorylation sites are outside the repeats they have only a weak influence on binding, whereas the phosphorylation at Ser262 inside the repeats inhibits binding and makes microtubules dynamically unstable. This site can be phosphorylated by kinases present in brain tissue, and it is uniquely phosphorylated in Alzheimer brain.