A J(CH)-modulated 2D (HACACO)NH pulse scheme for quantitative measurement of (13)C(alpha)-(1)H(alpha) couplings in (15)N, (13)C-labeled proteins.
A triple-resonance pulse sequence is presented for the quantitative measurement of (1)H(alpha)-(13)C(alpha) single-bond couplings in (15)N, (13)C uniformly labeled proteins. This (1)J(CH)-modulated (HACACO)NH experiment yields (1)H(N)-(15)N-correlated 2D spectra in which the amplitude of each peak is modulated by the (1)H(alpha)-(13)C(alpha) J coupling of the preceding residue, (i - 1). The experiment is demonstrated on a 1.0 mM sample of Rho130, the 15-kDa RNA binding domain of E. coli Rho factor. The average error in the measured coupling constants was less than 0.8%. This sequence allows the measurement of the (1)J(CH )couplings from a proton-nitrogen HSQC without the need for assigning the H(alpha) and C(alpha) resonances.
Hitchens
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Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.
McCallum
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Rule
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D002244:Carbon / Q000737:chemistry; D002247:Carbon Isotopes; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009584:Nitrogen / Q000737:chemistry; D009587:Nitrogen Isotopes; D012234:Rho Factor / Q000737:chemistry*; D012680:Sensitivity and Specificity
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012234:Rho Factor; D002244:Carbon; D006859:Hydrogen; D009584:Nitrogen