Accepting its Random Coil Nature Allows a Partial NMR Assignment of the Neuronal Tau Protein Caroline Smet, [a] Arnaud Leroy, [a, b] Alain Sillen, [a] Jean-Michel Wieruszeski, [a] Isabelle Landrieu, [a] and Guy Lippens* [a] Introduction The physiological function of the neuronal Tau protein involves the dynamic stabilization of tubulin into microtubules. [1, 2] Hence it plays an important role in neuronal development and plasticity. Moreover, the same microtubule-associated Tau pro- tein is one of the major components of the neurofibrillary paired helical fragments (PHFs) characteristic of Alzheimer’s disease (AD). [3, 4] Primarily composed of Tau, these tangles accu- mulate intracellularly in the diseased neurons, and their pres- ence correlates rather well with the cognitive impairments that result from the disease. [5] Despite its enormous biological and medical relevance, little is known about the detailed structure of Tau in solution. Small- angle X-ray scattering (SAXS) and other spectroscopic tech- niques such as circular dichroism (CD) and infrared spectros- copy (IR) have lead to a description of the protein as a random coil or Gaussian polymer. [6, 7] However, as these techniques only give an overall view of the global structure, local elements of secondary and/or tertiary structure might well escape observa- tion. The absence of a stable tertiary structure precludes the use of X-ray crystallography, but, as an alternative, NMR spectroscopy might well give hints about otherwise undetect- ed sequence specific structural elements. Although this latter technique has been successfully applied in the case of several other unfolded proteins, [8–10] the literature contains no detailed report about the NMR characterization of Tau. We have recently shown that proline residues and their dis- tinct influence on the random coil chemical-shift values can be used to assign at least those residues in Tau that precede a proline amino acid. [11] In order to extend the assignment to all residues irrespective of their direct neighbours, we demon- strate here the utility of a novel graphical method to handle the huge information content in the triple-resonance spectra of Tau. Pushing the comparison of the spectra of full-length adult Tau and its shorter foetal isoform to the limit of a com- parison with natural-abundance spectra of short peptides, allows us to proceed further in the assignment process. We demonstrate the assignment of Tau to such a level that i) the presence or absence of well-defined structural elements can be ascertained, and ii) the interaction with other molecular partners such as tubulin or aggregating agents can be studied. Results Conventional assignment strategies that use triple resonance experiments, correlate the Ca, CO or Cb of a given residue with that of its immediate neighbour. [12] A typical example of this strategy is the use of HNCA and HN(CO)CA experiments, where the Ca of residue [i] is seen from its own [i] amide cross peak in the first experiment and from the amide correlation of the next residue [i+1] in the HN(CO)CA. When applied to the full-length Tau protein, spectral overcrowding that results from i) the sheer size of the protein (Figure 1), ii) the limited chemi- cal-shift dispersion that results from the unfolded nature of the protein and iii) the high degeneracy in the primary sequence (Table 1) all made this strategy largely unsuccessful. The only nucleus that gives a good chemical-shift dispersion is the nitro- gen, as can be seen from the HSQC spectrum (Figure 2). We have previously observed that all Glycine Ca carbons, which represent a total of 49 residues (Table 1), fall within 0.5 ppm of their random-coil value. [13] We found the same extreme de- [a] C. Smet, Dr. A. Leroy, Dr. A. Sillen, Dr. J.-M. Wieruszeski, Dr. I. Landrieu, Dr. G. Lippens CNRS—UniversitØ de Lille 2, UMR 8525, Institut Pasteur de Lille B.P. 245, 59019 Lille Cedex (France) Fax: (+ 33) 320-871-233 E-mail: guy.lippens@pasteur-lille.fr [b] Dr. A. Leroy Laboratoire de Biochimie AppliquØe FacultØ de Pharmacie à Châtenay-Malabry (Paris XI) Tour D4 2 me Øtage, 5 rue Jean-Baptiste ClØment 92296 Chatenay-Malabry Cedex (France) A combined strategy to obtain a partial NMR assignment of the neuronal Tau protein is presented. Confronted with the extreme spectral degeneracy that the spectrum of this 441 amino acid long unstructured protein presents, we have introduced a graphi- cal procedure based on residue type-specific product planes. Combining this strategy with the search for pairwise motifs, and combining the spectra of different Tau isoforms and even of pep- tides derived from the native sequence, we arrive at a partial assignment that is sufficient to map the interactions of Tau with its molecular partners. The obtained assignments equally confirm the absence of regular secondary structure in the isolated pro- tein. ChemBioChem 2004, 5, 1639 – 1646 DOI: 10.1002/cbic.200400145 # 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1639