Correlation of backbone amide and side-chain (13)C resonances in perdeuterated proteins.
Side-chain carbon resonance assignments are difficult to obtain for larger proteins. While standard methods require protons for excitation and detection of magnetization, their presence is often unacceptable and often leads to unacceptable relaxation losses at the directly bound carbon sites. In this paper, pulse sequences are presented which provide connectivities between aliphatic side-chain (13)C and amide (1)H and (15)N chemical shifts in fully deuterated, (13)C/(15)N-enriched proteins. Magnetization either starts off from carbons or from both nitrogens and protons and is passed along the side-chain via (13)C-(13)C isotropic mixing. Direct rather than (13)CO-relayed (15)N-->(13)C(alpha) or (13)C(alpha)-->(15)N transfer steps allow the detection of intraresidual as well as sequential correlations. To avoid ambiguities between these two types in the three-dimensional version of the experiments, a fourth dimension can be introduced to achieve their separation along a (13)C(alpha) frequency axis. The novel methods are demonstrated with the uniformly (2)H/(13)C/(15)N labeled 35-kDa protein diisopropylfluorophosphatase from Loligo vulgaris.
Löhr
Frank
F
Institut für Biophysikalische Chemie, Johann Wolfgang Goethe-Universität Frankfurt, Biozentrum N230, Marie Curie-Strasse 9, Frankfurt am Main, 60439, Germany. murph@bpc.uni-frankfurt.de
Rüterjans
Heinz
H
D002247:Carbon Isotopes; D003903:Deuterium; D006859:Hydrogen; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D008280:Magnetics; D009587:Nitrogen Isotopes; D011506:Proteins / Q000737:chemistry*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D006859:Hydrogen; D003903:Deuterium