Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments

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Authors

MOTÁČKOVÁ Veronika NOVÁČEK Jiří ZAWADZKA-KAZIMIERCZUK Anna KAZIMIERCZUK Krzysztof ŽÍDEK Lukáš ŠANDEROVÁ Hana KRÁSNÝ Libor KOZMINSKI Wiktor SKLENÁŘ Vladimír

Year of publication 2010
Type Article in Periodical
Magazine / Source Journal of Biomolecular NMR
MU Faculty or unit

Faculty of Science

Citation
Web http://www.springerlink.com/content/?k=doi%3a%2810.1007%2fs10858-010-9447-3%29
Field Biochemistry
Keywords Unfolded proteins; Repetitive sequence; Multi-dimensional NMR; Non-uniform sampling; Assignment
Description 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, delta 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.
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