HCN, a triple-resonance NMR technique for selective observation of histidine and tryptophan side chains in 13C/15N-labeled proteins

James L. Sudmeier, Elissa L. Ash, Ulrich L. Günther, Xuelian Luo, Peter A. Bullock, William W. Bachovchin

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22 Scopus citations


HCN, a new 3D NMR technique for stepwise coherence transfer from 1H to 13C to 15N and reverse through direct spin couplings 1JCH and 1JCH, is presented as a method for detection and assignment of histidine and tryptophan side-chain 1H, 13C, and 15N resonances in uniformly 13C/15N-labeled proteins. Product-operator calculations of cross-peak volumes vs adjustable delay τ3 were employed for determination of optimal τ3. For the phosphatidylinositol 3-kinase (PI3K SH3 domain, MW = 9.6 kD ) at pH 6, H(C)N, the 1H/15N projection, produced observable cross peaks within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded welldefined cross peaks in 20 h for the 13C/15N-labeled origin-specific DNA binding domain from simian virus 40 T-antigen (T-ag-OBD131-259, MW = 15.4 kD) at pH 5.5. Resonances from all six histidines in T-ag-OBD were observed, and 11 of the 12 1H and 13C chemical shifts and 10 of the 12 15N chemical shifts were determined. The 13C dimension proved essential in assignment of the multiply overlapping 1H and 15N resonances. From the spectra recorded at a single pH, three of the imidazoles were essentially neutral and the other three were partially protonated (22-37%). HCN yielded strong cross peaks after 18 h on a 2.0 mM sample of phenylmethanesulfonyl fluoride (PMSF)-inhibited α-lytic protease (MW = 19.8 kD ) at pH 4.4. No spectra have been obtained, however, of native or boronic acid-inhibited α-lytic protease after 18 h at various temperatures ranging from 5 to 55°C, probably due to efficient relaxation of active-site imidazole 1H and/or 15N nuclei.

Original languageEnglish (US)
Pages (from-to)236-247
Number of pages12
JournalJournal of Magnetic Resonance - Series B
Issue number3
StatePublished - 1996

ASJC Scopus subject areas

  • Engineering(all)


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