title: Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments.
abstract: A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high dimensionality and long evolution times, allowed by non-uniform sampling in the indirect dimensions. Random distribution of the data points and Sparse Multidimensional Fourier Transform processing were used. Successful application of the assignment procedure to a particularly difficult protein, δ subunit of RNA polymerase from Bacillus subtilis, is shown to prove the efficiency of the strategy. The studied protein contains a disordered C-terminal region of 81 amino acids with a highly repetitive sequence. While the conventional assignment methods completely failed due to a very small differences in chemical shifts, the presented strategy provided a complete backbone and side-chain assignment.
authors.affiliation: National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Motáčková
authors.forename: Jiří
authors.initials: J
authors.lastname: Nováček
authors.forename: Anna
authors.initials: A
authors.lastname: Zawadzka-Kazimierczuk
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Kazimierczuk
authors.forename: Lukáš
authors.initials: L
authors.lastname: Zídek
authors.forename: Hana
authors.initials: H
authors.lastname: Sanderová
authors.forename: Libor
authors.initials: L
authors.lastname: Krásný
authors.forename: Wiktor
authors.initials: W
authors.lastname: Koźmiński
authors.forename: Vladimír
authors.initials: V
authors.lastname: Sklenář
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D003903:Deuterium; D005583:Fourier Analysis; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D003903:Deuterium