A functional role for intrinsic disorder in the tau-tubulin complex

Abstract

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

Insights into tau function and dysfunction through single-molecule fluorescence

“…The protocol developed in our lab for the expression and purification of tau has been used for both full-length and truncated forms of the protein (Elbaum-Garfinkle et al, 2014, 2010; Elbaum-Garfinkle & Rhoades, 2012; Li et al, 2015; Melo et al, 2016). We cloned tau into a pET—HT plasmid with an N-terminal His-tag and a tobacco etch virus cleavage site.…”

“…We cloned tau into a pET—HT plasmid with an N-terminal His-tag and a tobacco etch virus cleavage site. Expression and purification are performed as published previously (Elbaum-Garfinkle et al, 2014; Elbaum-Garfinkle & Rhoades, 2012; Melo et al, 2016), with nickel-affinity and size-exclusion chromatography steps. All chromatography is carried out at 4°C to minimize protein degradation.…”

“…The protocol described later for labeling tau with donor and acceptor fluorophores was developed to minimize the fraction of donor-only labeled protein that contributes to the ET eff =0 peak in smFRET histograms (Melo et al, 2016). Our most commonly used donor and acceptor fluorophores are Alexa Fluor 488 and Alexa Fluor 594 maleimide, respectively.…”

“…There are numerous experimental challenges to achieving this goal, including tau’s intrinsically disordered structure (Cleveland, Hwo, & Kirschner, 1977; Schweers, Schonbrunn-Hanebeck, Marx, & Mandelkow, 1994), its propensity to aggregate (Chirita, Congdon, Yin, & Kuret, 2005; Friedhoff, Schneider, Mandelkow, & Mandelkow, 1998), and the dynamic nature of microtubule polymerization (Drechsel, Hyman, Cobb, & Kirschner, 1992; Trinczek, Biernat, Baumann, Mandelkow, & Mandelkow, 1995). To overcome these limitations, our lab applies single-molecule fluorescence techniques, namely fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) to characterize its aggregation prone structures (Elbaum-Garfinkle, Ramlall, & Rhoades, 2010; Elbaum-Garfinkle & Rhoades, 2012) and its binding to tubulin, an important aspect of microtubule polymerization (Elbaum-Garfinkle, Cobb, Compton, Li, & Rhoades, 2014; Li, Culver, & Rhoades, 2015; Melo et al, 2016). In this chapter, we describe our protocols and technical aspects of the application of FCS and smFRET to study tau and its interactions with binding partners.…”

“…It can provide unique insights into the conformations of large and dynamic proteins—such as tau—where higher resolution structural approaches cannot be applied (Elbaum-Garfinkle & Rhoades, 2012; Melo et al, 2016). FRET is a nonradiative transfer of energy from a donor fluorophore to an acceptor fluorophore that occurs through a dipole–dipole mechanism.…”