Assignment of uniformly carbon-13-enriched proteins and optimization of their carbon lineshapes

The purpose of this work was to establish a reliable methodology for the assignment of solid-state NMR spectra of uniformly 13C, 15N-enriched proteins. Success of an assignment procedure critically depends on the resolution of multidimensional solid-state NMR spectra, while the resolution is mostly determined by 13C linewidths. First, we attempted to dissect, analyze, and control the contributions of J-coupling and residual homo-nuclear dipolar coupling interactions to the carbon linewidths of a model compound, uniformly 13C,15N-enriched crystalline alanine. We established both experimentally and computationally that, at moderate magnetic field strengths and spinning frequencies, the major and comparable contributors to the carbon linewidths are 13C- 13C J-coupling interactions and the second-order dipolar shift. These contributions were separated spectroscopically using a homo-nuclear J-decoupling sequence, the performance of which we fully characterized for two chosen spinning frequencies. A combination of the second-order dipolar shift and J-coupling interactions gave rise to characteristic 13C triplet lineshapes, which we observed initially in the spectra of alanine and then in the spectra of microcrystalline uniformly 13C, 15N-enriched ubiquitin. We showed that the resolution in two-dimensional spectra of alanine and ubiquitin could be significantly improved by using J-decoupling.; Next, we report carbon and nitrogen assignments for the majority of sites in uniformly 13C,15N-enriched ubiquitin. Sequential assignments were carried out using a set of three-dimensional experiments recorded at 400 MHz, which provided intra-residue and sequential carbon and nitrogen correlations. To verify the spin system type and obtain additional assignments for the sidechain carbons, we used a 13C- 13C spin diffusion experiment recorded at 800 MHz. We found that the regions missing in the solid-state NMR protein spectra correlate with the regions of increased backbone mobility as attested by the elevated values of temperature factors in the crystal structure of ubiquitin and by the depressed values of backbone order parameters obtained in solution NMR experiments. The solid-state NMR chemical shifts were then compared with the solution NMR values, and the perturbed residues were identified. To our knowledge, ubiquitin is the second protein assigned to this degree of completeness using solid-state NMR methods.