Automatic Assignment of the Intrinsically Disordered Protein Tau with 441-Residues

Abstract

Abstract: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 sevendimensional 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.In the past decade intrinsically disordered proteins (IDPs) obtained significant attention as more than 30% of eukaryotic proteins comprise unstructured regions larger than 50 consecutive residues. 1 A hallmark of IDPs is the lack of a well-structured threedimensional fold. 2 Despite the absence of a rigid secondary or tertiary structure, IDPs carry out many important functions in the cell, for example the regulation of transcription and translation, the storage of small molecules, and the regulation of the self-assembly of large multiprotein complexes. 3 Tau, a prototype of IDPs, binds to microtubules and plays a key role in Alzheimer disease. 4 Since disordered proteins tend to be highly flexible and have variable conformations, they have mostly not been amenable for structure analysis by crystallography. Thus NMR spectroscopy is the only method that allows a description of their conformations and dynamics with high resolution. 5 The lack of an ordered structure, however, causes dramatic signal overlap limiting previous NMR investigations of IDPs to around 200 residues (e.g., refs 6, 7). For the largest Tau isoform with 441 residues, htau40 ( Figure 1), the number of overlapping signals is 3.5-fold higher than that in the largest currently assigned globular protein. 8 In addition, the large number of proline residues, the strongly repetitive primary sequence in the repeat domains, and the complexity of the sequence further complicate the resonance assignment. 8,9 Only as part of an ongoing project taking several months to years and building on the assignment of smaller fragments of Tau in a "divide-andconquer" strategy, we were recently able to assign the backbone resonances of the full-length protein. 8 Here we demonstrate that the resonance assignment of 441-residue Tau and its smaller isoforms, htau24 (383 residues) and htau23 (352 residues) (Figure 1), 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.Due to the lack of stable hydrogen bonds, NMR spectra of IDPs are sensitive to solvent exchange. At pH 6.8 an increase in temperature from 5 to 35°C results in the broadening of ab...

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