A novel approach for sequential assignment of proton, carbon-13, and nitrogen-15 spectra of larger proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin

Mitsuhiko Ikura, Lewis E. Kay, Ad Bax
1990 Biochemistry  
A novel approach is described for obtaining sequential assignment of the backbone 'H, I3C, and 15N resonances of larger proteins. The approach is demonstrated for the protein calmodulin (16.7 kDa), uniformly (-95%) labeled with 15N and 13C. Sequential assignment of the backbone residues by standard methods was not possible because of the very narrow chemical shift distribution range of both NH and CaH protons in this largely a-helical protein. We demonstrate that the combined use of four new
more » ... use of four new types of heteronuclear 3D N M R spectra together with the previously described HOHAHA-HMQC 3D experiment Biochemistry 28, 6150-61 561 can provide unambiguous sequential assignment of protein backbone resonances. Sequential connectivity is derived from one-bond J couplings and the procedure is therefore independent of the backbone conformation. All the new 3D N M R experiments use I Abbreviations: INEPT, insensitive nuclei enhanced by polarization transfer; HCACO, proton to a-carbon to carbonyl correlation; HCA-(CO)N, proton to a-carbon (via carbonyl) to nitrogen correlation; HMQC, heteronuclear multiple quantum correlation; HNCO, amide proton to nitrogen to carbonyl correlation; HNCA, amide proton to nitrogen to a-carbon correlation; HOHAHA, homonuclear Hartmann-Hahn; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; 2D, two-dimensional; 3D, three-dimensional.
doi:10.1021/bi00471a022 pmid:2372549 fatcat:qi2xbaq7zrbxhfhts7ui2gjwbq