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

FEBS Letters

Lubienski, Michael J.¹  Jones, David N.M.¹  Fersht, Alan R.¹  Bycroft, Mark¹ 

[1] MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK

关键词: Barnase-barstar complex;    NMR assigment;    NMR;    nuclear magnetic resonance;    2D;    two-dimensional;    3D;    three-dimensional;    NOE;    nuclear Overhauser enhancement;    NOESY;    two-dimensional NOE-correlated spectroscopy;    TOCSY;    total correlated spectroscopy;    HMQC;    2D 1H-detected heteronuclear multiple-quantum correlation;    HSQC;    2D 1H-detected heteronuclear single-quantum correlation;   

DOI  :  10.1016/0014-5793(93)80319-P

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown