J. Biochem, 1990, Vol. 108, No. 2 149-153
© 1990 Japanese Biochemical Society
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Combination of Heteronuclear 1H-15 and 1H-13C Three-Dimensional Nuclear Magnetic Resonance Experiments for Amide-Directed Sequential Assignment in Larger Proteins
*Biometrology Lab, JEOL Ltd. Ahishkna, Tokyo 196
**Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd. Machida, Tokyo 194
***Protein Engineering Research Institute Suita, Osaka 565
1To whom correspondence should be addressed
A new strategy for the sequential assignment of backbone proton resonances in larger proteins Involving a unique combination of four types of heteronuclear three-dimensional (3D) NMR spectroseopies is reported. This method relies on the uniform labeling of amide nitrogens with 15N and of 
-carbons with 13C. Heteronuclear 1H-15 TOCSY-HMQC and NOESY-HMQC experiments can reveal connections between cross-peaks arising from the NH1-CHt=1 and NH1-CHt=1 connectivities in the finger-print region in general. They also specifically reveal the sequential amide-amide connectivities among the amide cross-peaks for the -helices. Heteronuclear 1H-13C HMQC-TOCSY and HMQC-NOESY experiments can reveal connections between cross-peaks arising from the NH1-C H and NH1-Ct=1H con nectivities In the finger-print region in general. The combination of the two sets of results reveals the complete unambiguous sequential connection of cross-peaks for the proton resonances in the peptide backbone. The application of the new strategy is reported for a protein, ribonuclease H, with a molecular weight of 17.6 kDa.