Measurement of residual dipolar couplings from 1Halpha to 13Calpha and 15N using a simple HNCA-based experiment.
Novel NMR pulse schemes for simultaneous measurement of 1D(C)alpha(H)alpha and 2D(NH)alpha residual dipolar couplings in proteins is presented. We show that 2D(NH)alpha coupling can be very useful for protein structure determination. The 2D(NH)alpha coupling can be measured from 15N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large 1D(C)alpha(H)alpha coupling. The dynamic range of 2D(NH)alpha coupling is comparable to 1D(NC') coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on 15N/13C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both 1D(C)alpha(H)alpha and 2D(NH)alpha couplings, was obtained.
D002247:Carbon Isotopes; D003460:Crystallization; D006801:Humans; D006859:Hydrogen; D008280:Magnetics; D008962:Models, Theoretical; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D012996:Solutions; D025801:Ubiquitin / Q000737:chemistry
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D012996:Solutions; D025801:Ubiquitin; D006859:Hydrogen