Assignments of Carbon NMR Resonances for Microcrystalline Ubiquitin

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Abstract
Solid-state NMR 2D spectroscopy was used to correlate carbon backbone and side-chain chemical shifts for uniformly 13C,15N-enriched microcrystalline ubiquitin. High applied field strengths, 800 MHz for protons, moderate proton decoupling fields, 80−100 kHz, and high magic angle sample spinning frequencies, 20 kHz, were used to narrow the most of the carbon line widths to 0.5−0.8 ppm. Homonuclear magnetization transfer was effected by matching the proton RF field to the spinning frequency, the so-called dipolar-assisted rotational resonance (DARR) (Takegoshi, K.; Nakamura, S.; Terao, T. Chem. Phys. Lett.2001, 344, 631−637), and a mixing time of 20 ms was used to maximize the intensity of one-bond transfers between carbon atoms. This polarization transfer sequence resulted in roughly 14% transfer efficiencies for directly bonded carbon pairs and 4% transfer efficiencies for carbons separated by a third carbon. With this simple procedure, the majority of the one-bond correlations was observed with moderate transfer efficiencies, and many two-bond correlations were also observed with weaker intensities. Spin systems could be identified for more than half of the amino acid side chains, and site-specific assignments were readily possible via comparison with 400 MHz 15N−13C−13C correlation spectroscopy (described separately).