5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion
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
- Dunker AK, Obradovic Z, Romero P, Garner EC, Brown CJ (2000) Intrinsic protein disorder in complete genomes. Genome Inform 11:161–171Google Scholar
- Eliezer D (2007) Characterizing residual structure in disordered protein states using nuclear magnetic resonance. Methods Mol Biol 350:49–67Google Scholar
- Motáčková V, Šanderová H, Žídek L, Nováček J, Padrta P, Švenková A, Korelusová J, Jonák J, Krásný L, Sklenář V (2010) Solution structure of the N-terminal domain of Bacillus subtilis delta subunit of RNA polymerase and its classification based on structural homologs. Proteins Struct Funct Bioinf 78(7):1807–1810Google Scholar