Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex.

Staphylococcal nuclease H124L is a recombinant protein produced in Escherichia coli whose sequence is identical with that of the nuclease produced by the V8 variant of Staphylococcus aureus. The enzyme-metal ion activator-nucleotide inhibitor ternary complex, nuclease H124L-thymidine 3{prime},5{prime}-bisphosphate-Ca{sup 2+}, was investigated by two-dimensional (SD) NMR techniques. Efficient overproduction of the enzyme facilitated the production of random fractionally deuterated protein, which proved essential for detailed NMR analysis. {sup 1}H NMR spin systems were analyzed by conventional 2D {sup 1}H({sup 1}H) methods: COSY, relayed COSY, HOHAHA, and NOESY. Assignments obtained by {sup 1}H NMR experiments were confirmed and extended by {sup 1}H-{sup 13}C and {sup 1}H-{sup 15}N heteronuclear NMR experiments. Spectra of the ternary complexes prepared with protein at natural abundance and at 50% random fractional deuteration provided the information needed for sequence-specific assignments of 121 of the 149 amino acid residues. Short- and intermediate-range NOE connectivities allowed the determination of secondary structural features of the ternary complex. The solution structure of this ternary complex shows a close correspondence to the crystal structure of the nuclease wt-thymidine 3{prime},5{prime}-bisphosphate-Ca{sup 2+} ternary complex.