Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehyde

Matthew W. Vetting, Subray S. Hegde, John S. Blanchard

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane-dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6 Å resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68 Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups.

Original languageEnglish (US)
Pages (from-to)462-469
Number of pages8
JournalActa Crystallographica Section D: Biological Crystallography
Volume65
Issue number5
DOIs
Publication statusPublished - Apr 18 2009

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Keywords

  • Chemical modification
  • Glutaraldehyde
  • Pentapeptide-repeat proteins
  • Reductive cyclic pentylation

ASJC Scopus subject areas

  • Structural Biology

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