Protonless 13C direct detection NMR: characterization of the 37 kDa trimeric protein CutA1

The major limitation of nuclear magnetic resonance spectroscopy arises from the increase of nuclear transverse relaxation rates with increasing molecular mass. This causes reduction in spectral resolution and coherence transfer efficiency. The use of 2H-labeling to eliminate 1H-mediated relaxation pathways and the constructive use of cross correlation effects (TROSY, CRINEPT) alleviate the phenomenon. An alternative approach is to use direct detection of heteronuclei. Specifically designed 13C direct detection experiments can complement the set of 1H-based NMR experiments commonly used for structure determination providing an additional source of information less affected by the detrimental transverse relaxation effect. We applied this novel methodology to the study of the CutA1 protein (12.3 kDa) from E. coli that forms a homotrimer in solution with a total molecular mass of 37 kDa. In this work we demonstrate that the information available from 13C direct detection experiments makes it possible to completely assign the NMR resonances of the backbone of this 37 kDa trimeric protein without the need of deuteration. The structural and dynamical knowledge obtained for this system may contribute to understand its biological role.