title: Efficient protocol for backbone and side-chain assignments of large, intrinsically disordered proteins: transient secondary structure analysis of 49.2 kDa microtubule associated protein 2c.
abstract: Microtubule-associated proteins (MAPs) are abundantly present in axons and dendrites, and have been shown to play crucial role during the neuronal morphogenesis. The period of main dendritic outgrowth and synaptogenesis coincides with high expression levels of one of MAPs, the MAP2c, in rats. The MAP2c is a 49.2 kDa intrinsically disordered protein. To achieve an atomic resolution characterization of such a large protein, we have developed a protocol based on the acquisition of two five-dimensional (13)C-directly detected NMR experiments. Our previously published 5D CACONCACO experiment (Nováček et al. in J Biomol NMR 50(1):1-11, 2011) provides the sequential assignment of the backbone resonances, which is not interrupted by the presence of the proline residues in the amino acid sequence. A novel 5D HC(CC-TOCSY)CACON experiment facilitates the assignment of the aliphatic side chain resonances. To streamline the data analysis, we have developed a semi-automated procedure for signal assignments. The obtained data provides the first atomic resolution insight into the conformational state of MAP2c and constitutes a model for further functional studies of MAPs.
authors.affiliation: Faculty of Science, NCBR, and CEITEC, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Nováček
authors.forename: Lubomír
authors.initials: L
authors.lastname: Janda
authors.forename: Radka
authors.initials: R
authors.lastname: Dopitová
authors.forename: Lukáš
authors.initials: L
authors.lastname: Žídek
authors.forename: Vladimír
authors.initials: V
authors.lastname: Sklenář
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D005998:Glycine; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D051381:Rats
chemical_list: D064267:Intrinsically Disordered Proteins; D008869:Microtubule-Associated Proteins; D005998:Glycine