SOLUTION STUDIES OF STAPHYLOCOCCAL NUCLEASE H124L .1. BACKBONE H-1 AND N-15 RESONANCES AND SECONDARY STRUCTURE OF THE UNLIGATED ENZYME AS IDENTIFIED BY 3-DIMENSIONAL NMR-SPECTROSCOPY

The backbone H-1 and N-15 resonances of unligated staphylococcal nuclease H124L (recombinant protein produced in Escherichia coli whose sequence is identical to the nuclease produced by the V8 strain of Staphylococcus aureus) have been assigned by three-dimensional (3D) H-1-N-15 NOESY-HMQC NMR spectroscopy at 14.1 tesla. The protein sample used in this study was labeled uniformly with N-15 to a level greater than 95% by growing the E. coli host on a medium containing [99% N-15]ammonium sulfate as the sole nitrogen source. The assignments include 82% of the backbone H-1(N) and H-1(alpha) resonances as well as the N-15 resonances of non-proline residues. Secondary structural elements (alpha-helices, beta-sheets, reverse turns, and loops) were determined by analysis of patterns of NOE connectivities present in the 3D spectrum.