Tau is an intrinsically disordered protein with an important role in maintaining the dynamic instability of neuronal microtubules. Despite intensive study, a detailed understanding of the functional mechanism of tau is lacking. Here, we address this deficiency by using intramolecular single-molecule Förster Resonance Energy Transfer (smFRET) to characterize the conformational ensemble of tau bound to soluble tubulin heterodimers.Tau adopts an open conformation on binding tubulin, in which the long-range contacts between both termini and the microtubule binding region that characterize its compact solution structure are diminished. Moreover, the individual repeats within the microtubule binding region that directly interface with tubulin expand to accommodate tubulin binding, despite a lack of extension in the overall dimensions of this region. These results suggest that the disordered nature of tau provides the significant flexibility required to allow for local changes in conformation while preserving global features. The tubulin-associated conformational ensemble is distinct from its aggregation-prone one, highlighting differences between functional and dysfunctional states of tau. Using constraints derived from our measurements, we construct a model of tubulin-bound tau, which draws attention to the importance of the role of tau's conformational plasticity in function.single-molecule FRET | intrinsically disordered proteins | microtubuleassociated protein | tauopathies | Alzheimer's disease
Tubulin, the building block of microtubules, is subject to chemically diverse and evolutionarily conserved post-translational modifications that mark microtubules for specific functions in the cell. Here we describe in vitro methods for generating homogeneous acetylated, glutamylated, or tyrosinated tubulin and microtubules using recombinantly expressed and purified modification enzymes. The generation of differentially modified microtubules now enables a mechanistic dissection of the effects of tubulin posttranslational modifications on the dynamics and mechanical properties of microtubules as well as the behavior of motors and microtubule associated proteins.
Bundles of taxol-stabilized microtubules (MTs)-hollow protein nanotubes comprised of assembled ab-tubulin heterodimers-spontaneously assemble above a critical concentration of tetravalent spermine and are stable over long times at room temperature. Here we report that at concentrations of spermine several-fold higher the MT bundles (B MT) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (B ITT), the outside surface of which corresponds to the inner surface of the B MT tubules. Using transmission electron microscopy and synchrotron small-angle X-ray scattering, we quantitatively determined both the nature of the B MT-to-B ITT transformation pathway and the structure of the B ITT phase. Inverted tubulin tubules provide a platform for studies requiring exposure and availability of the inside, luminal surface of MTs to MT-targeted drugs and MTassociated proteins. (ref.
Sterols, vital and abundant components of plasma membranes, are involved in numerous cellular biochemical processes such as regulation of membrane properties and signaling events. However, these sterol interactions are not completely elucidated at the atomic level; this impedes further investigations into disorders related to these biological activities. Combining solid-state NMR (SSNMR), high yield biosynthesis of isotopically labeled sterols, and molecular dynamics, we aim to understand the key interactions that determine the sterol-specificity to amphotericin B (AmB), a powerful but toxic antifungal drug used to treat life-threatening fungal infections. AmB forms large aggregates that are neither crystalline nor soluble, making structural information difficult or impossible to obtain via x-ray crystallography or solution-state NMR. We have recently determined that AmB kills yeast cells primarily by binding to ergosterol 1 (Erg) and forming a large extramembranous sterol sponge. 2 These findings were enabled by the biosynthesis of fractionally 13 C labeled Erg and state-of-the-art SSNMR techniques. The sterol sponge model hypothesizes that interactions of AmB with Erg determine its ability to kill yeast, whereas binding to cholesterol is responsible for determining its toxicity in human cells. SSNMR spectroscopy is uniquely able to detect and quantify the binding of sterols to AmB in the sterol sponge in atomistic detail. These studies provide a roadmap towards an improved therapeutic index 3 for this drug while simultaneously elucidating additional details about the molecular mechanism of AmB-sterol interactions. (1)''Amphotericin primarily kills yeast by simply binding ergosterol'', Gray et al.
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