5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion
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Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of 13C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.
KeywordsIntrinsically disordered proteins Non-uniform sampling 13C detection Longitudinal relaxation optimization Backbone assignment
This work was supported by the Grants of the Ministry of Education of Czech Republic MSM0021622413 and LC06030, by the Grants 204/09/0583, 301/09/H004 and P206/11/0758 from Czech Science Foundation, by the EU/ grant POSTBIOMIN (FP7-REGPOT-2007-1 No. 205872), by MPD program from Foundation for Polish Sciences that was co-financed by the European Regional Development Fund. Financial support including the form of Access to the Bio-NMR Research Infrastructure co-funded under the 7th Framework Programme of the EC (FP7/2007-2013) grant agreement 261863 for conducting the research is gratefully acknowledged.
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