Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen-deuterium exchange.
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 characterized 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.
Jones
D N
DN
Department of Chemistry, University of Cambridge, UK.
Bycroft
M
M
Lubienski
M J
MJ
Fersht
A R
AR
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; C057438:barstar protein, Bacillus amyloliquefaciens; D006859:Hydrogen; D003903:Deuterium; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease