Histidine 121 of staphylococcal nuclease. Correction of the Hδ2 1H NMR assignment and reinterpretation of the role this residue plays in conformational heterogeneity of the protein

Heteronuclear two-dimensional NMR studies of wild-type staphylococcal nuclease containing histidine residues uniformly labeled with carbon-13 (26% isotope) have led to full analysis of the aromatic parts of the histidine {sup 1}H and {sup 13}C spin systems. The {sup 1}H{sup {delta}2} and {sup 13}C{sup {delta}2} resonances of His{sup 121} were found to be split as the result of the N{sub al} {r equilibrium} N{sub a2} conformational equilibrium described previously and attributed to cis-trans isomerism about the Lys{sup 116}-Pro{sup 117} peptide bond (N{sub al}, cis; N{sub a2}, trans). The relative intensities of the pair of {sup 1}H{sup {epsilon}1} peaks from the same residue. A soluble-mutant enzyme (nuclease G79S + H124L), which exhibited a drastically altered (N{sub a1}) to (N{sub a2}) ratio, provided additional evidence that the pair of {sup 1}H{sup {delta}2} peaks and the pair of {sup 1}H{sup {epsilon}1} peaks of His{sup 121} report on the same conformational equilibrium (N{sub a1} {r equilibrium} N{sub a2}). The unusual chemical shift of the {sup 1}H{sup {delta}2} of His{sup 121} is attributed to diamagnetic shielding by the aromatic ring of Tyr{sup 91} as verified by ring-current calculations based on two X-ray structures for wild-type staphylococcal nuclease.