Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen‐deuterium exchange

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Abstract
The extracellular ribonuclease from Bacillus amyloliquifaciens, barnase, forms a tightly‐bound one‐to‐one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was characterised by comparing the differences in the chemical shift and hydrogen‐deuterium exchange rates between free and bound barnase. Chemical shift assignments of barnase in the complex with barstar were determined from 3D NOESY‐HMQC and TOCSY‐HMQC spectra of a complex that had been prepared with uniformly 15N‐labelled barnase and unlabelled barstar. Hydrogen exchange rates were obtained from an analysis of a series of [15N]HMQC spectra of a sample prepared in the same manner exchanged into D2O. The largest changes in either chemical shift or hydrogen‐deuterium exchange rate are observed for residues located in the active‐site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site.