NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states

Abstract

NMR signals from all four histidine ring C epsilon protons and three of the four histidine C delta protons in the protein staphylococcal nuclease have been assigned by comparing spectra of the wild-type (Foggi strain) protein to spectra of three variants that each lack a different histidine residue. All proteins studied were cloned and overproduced in Escherichia coli. The NMR spectra of the three mutant proteins (H8R, H46Y, and H124L) used to make these assignments were similar to one another and to those of the wild type, except for signals from the mutated residues. The pKa values of those histidines conserved between the wild type and the mutants remained essentially unchanged. Multiple histidine C epsilon proton resonances due to non-native forms of nuclease were observed in both thermally induced and acid-induced unfolding. Residue-specific assignments of H epsilon protons in the thermally denatured forms of the mutant H46Y were obtained from connectivities to the native state by saturation transfer.

Stress and strain in staphylococcal nuclease

“…The presence of multiple resonances for each histidine HE' proton indicates that the K116G substitution has maintained the structural heterogeneity observed in nuclease A. The chemical shifts of the four sets of resonances are sufficiently similar to those of the nuclease A spectrum that the wild-type sequence assignments can be inferred (Alexandrescu et al, 1988;Kautz et al, 1990), but the relative chemical shifts within each pair of resonances are significantly perturbed so that assignment of the cis and trans states cannot be made by comparison with nuclease A. For the three resonances where heterogeneity is displayed in nuclease A (H8, H124, H121) the major and minor resonances of K116G appear to be reversed in position when compared to wild-type protein (Fig.…”

Electrostatic Effects in a Network of Polar and Ionizable Groups in Staphylococcal Nuclease

“…58 The chemical shifts of the C ε1 H of each histidine are distinct and downfield from the majority of the resonances. They are clearly distinguishable over the course of a titration experiment.…”

Acid-induced Unfolding of the Amino-terminal Domains of the Lethal and Edema Factors of Anthrax Toxin

“…Similar depressed pK values for desolvated His residues, which destabilized the native state, have been observed in apomyoglobin [48][49][50][51] and staphylococcal nuclease. 44,[52][53][54] In apomyoglobin, the observed acidinduced transition from N/I is driven by a couple of His residues with very depressed pK values, 51 which may be considered "hot spots". 47,51,55 The alternative view that the loss of globally dispersed charge-charge interactions is responsible for the acid-denaturation is less likely, since such interactions are generally weaker.…”