HCN, a new 3D NMR technique for stepwise coherence transfer rarely sufficient to establish the mechanism through which from 1 H to 13 C to 15 N and reverse through direct spin couplings it participates in the protein's function. The structure may 1 J CH and 1 J CN , is presented as a method for detection and assignsuggest mechanisms, but final confirmation and understandment of histidine and tryptophan side-chain 1 H, 13 C, and 15 N resoing ultimately reduce to the chemistry of histidine as well nances in uniformly 13 C/ 15 N-labeled proteins. Product-operator as to its geometric positioning. Histidine chemistry includes calculations of cross-peak volumes vs adjustable delay t 3 were such factors as (i) its protonation state and solvent accessiemployed for determination of optimal t 3 . For the phosphatidylbility, as determined, for example, by whether it titrates inositol 3-kinase (PI3K SH3 domain, MW Å 9.6 kD) at pH 6, with changes in solvent pH, (ii) its tautomeric structure and H(C)N, the 1 H/ 15 N projection, produced observable cross peaks equilibration rates, (iii) its hydrogen-bonding status, and within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded well-(iv) how (i) to (iii) above are affected by relevant events defined cross peaks in 20 h for the 13 C/ 15 N-labeled origin-specific such as the binding of substrates, ligands, or inhibitors. De-DNA binding domain from simian virus 40 T-antigen (T-agtermination of these variables can substantially illuminate OBD 131 -259 , MW Å 15.4 kD) at pH 5.5. Resonances from all six the role of histidine and consequently enhance overall underhistidines in T-ag-OBD were observed, and 11 of the 12 1 H and standing of the parent molecule. Solved protein structures 13 C chemical shifts and 10 of the 12 15 N chemical shifts were are usually mute regarding the above chemical factors. determined. The 13 C dimension proved essential in assignment of Our past work has demonstrated that the behavior of the the multiply overlapping 1 H and 15 N resonances. From the spectra imidazole-ring 15 N NMR signals is the best probe into the recorded at a single pH, three of the imidazoles were essentially rich chemistry of histidine residues in proteins (1-5). Most