Abstract
The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET) assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using microtubules stabilized with Flutax-2 as donor and tau labeled with rhodamine as acceptor, a donor-to-acceptor distance of 54 ± 1 Å was found. A molecular model is proposed in which Flutax-2 is directly accessible to tau-rhodamine molecules for energy transfer. By titration, we calculated the stoichiometric dissociation constant to be equal to 1.0 ± 0.5 µM. The influence of the C-terminal tails of αβ-tubulin on the tau-microtubule interaction is presented once a procedure to form homogeneous solution of cleaved tubulin has been determined. The results indicate that the C-terminal tails of α- and β-tubulin by electrostatic effects and of recruitment seem to be involved in the binding mechanism of tau.
Highlights
Tau is the major microtubule associated protein (MAP) in neuronal axons [1] that stabilizes microtubules and maintains neuronal process [2,3]
Before looking at the energy transfer between this species and the microtubules stabilized with Flutax-2, which act as the donor, the functionality of tau-rhodamine to induce microtubules formation was studied
The distance separating the microtubules stabilized with Flutax-2 and tau derivatized with 5,6-TAMRA was found to be 54 ± 1 Å at 47% efficiency
Summary
Tau is the major microtubule associated protein (MAP) in neuronal axons [1] that stabilizes microtubules and maintains neuronal process [2,3]. In a previous paper as co-authors, our laboratory has already studied the interaction of the largest tau isoform (hTau40) with taxol stabilized microtubules using cosedimentation, resonance magnetic nuclear (RMN) and fluorescence techniques [11]. By cosedimentation and fluorescence resonance energy transfer (FRET), binding parameters were derived. FRET, typically, leads to a reduction in the donor’s fluorescence intensity and excited state lifetime and to an increase in acceptor’s emission intensity This technique is a useful tool to estimate distance between donor and acceptor fluorophores [16] and conformational changes in biomolecules [17]. To investigate the binding of tau protein to the microtubules, the energy transfer between microtubules stabilized with Flutax-2 and hTau protein labeled with carboxy tetramethylrhodamine succinimidyl ester was measured. Once experimental conditions to produce a homogeneous solution of cleaved tubulin were set up, binding parameters were measured and compared to those obtained with undigested tubulin
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