Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments.
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.
Motáčková
Veronika
V
National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic.
Nováček
Jiří
J
Zawadzka-Kazimierczuk
Anna
A
Kazimierczuk
Krzysztof
K
Zídek
Lukáš
L
Sanderová
Hana
H
Krásný
Libor
L
Koźmiński
Wiktor
W
Sklenář
Vladimír
V
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
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D003903:Deuterium