Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehyde.
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 A 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.
Vetting
Matthew W
MW
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. vetting@aecom.yu.edu
Hegde
Subray S
SS
Blanchard
John S
JS
D000588:Amines / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D001880:Boranes / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D004123:Dimethylamines / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D013293:Enterococcus faecalis / Q000737:chemistry*; D005976:Glutaral / Q000494:pharmacology*; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000187:drug effects; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020449:Repetitive Sequences, Amino Acid
D000588:Amines; D001426:Bacterial Proteins; D001880:Boranes; D003432:Cross-Linking Reagents; D004123:Dimethylamines; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D008239:Lysine; D005976:Glutaral