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 1H and 13C spin systems. The 1Hδ2 and 13Cδ2 resonances of His121 were found to be split as the result of the Na1 ⥨ NA2 conformational equilibrium described previously and attributed to cis-trans isomerism about the Lys116-Pro117 peptide bond (Na1 cis; Na2, trans)3 The relative intensities of the pair of 1Hδ2 peaks of His121 showed the same dependence on ligation at the active site as do the relative intensities of the pair of 1Hϵ1 peaks from the same residue. A double-mutant enzyme (nuclease G79S+H124L), which exhibited a drastically altered [Nal] to [Na2] ratio, provided additional evidence that the pair of 1Hδ2 peaks and the pair of 1Hϵ1 peaks of His121 report on the same conformational equilibrium (Nal ⥨ Na2). The unusual chemical shift of the 1Hδ2 of His121 is attributed to diamagnetic shielding by the aromatic ring of Tyr91 as verified by ring-current calculations based on two X-ray structures for wild-type staphylococcal nuclease.18 Since both the1Hδ2 and 13Cδ2 resonances showed large chemical shift changes on going from the Nal state (1Hδ2 = 4.3 ppm; 13Cδ2 =117 ppm) to the Na2 state (1Hδ2 = 4.9 ppm; 13Cδ2 = 121 ppm), the conformational change must alter the relative orientations of the His121 and Tyr91 rings. The results correct earlier misassignment of peaks around 6.3 ppm (at pH· 7.8) to 1Hδ2 of His121 along with misinterpretation of the effect of the Na1 ⥨ Na2 equilibrium on this residue.6.
ASJC Scopus subject areas
- Colloid and Surface Chemistry