Assignments of carbon NMR resonances for microcrystalline ubiquitin.
Solid-state NMR 2D spectroscopy was used to correlate carbon backbone and side-chain chemical shifts for uniformly (13)C,(15)N-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 (15)N-(13)C-(13)C correlation spectroscopy (described separately).
Igumenova
Tatyana I
TI
Department of Chemistry, Columbia University, 3000 Broadway MC 3113, New York, New York 10027, USA.
McDermott
Ann E
AE
Zilm
Kurt W
KW
Martin
Rachel W
RW
Paulson
Eric K
EK
Wand
A Joshua
AJ
D000596:Amino Acids / Q000737:chemistry; D002247:Carbon Isotopes; D003460:Crystallization; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D016011:Normal Distribution; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D012996:Solutions / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry*
D000596:Amino Acids; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D012996:Solutions; D025801:Ubiquitin