Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehyde
- Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)
A method to modify proteins with glutaraldehyde under reducing conditions is presented. Treatment with glutaraldehyde and dimethylaminoborane was found to result in cyclic pentylation of free amines and facilitated the structural determination of a protein previously recalcitrant to the formation of diffraction quality crystals. 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.
- OSTI ID:
- 22351175
- Journal Information:
- Acta Crystallographica. Section D: Biological Crystallography, Vol. 65, Issue Pt 5; Other Information: PMCID: PMC2672816; PMID: 19390151; PUBLISHER-ID: bw5278; OAI: oai:pubmedcentral.nih.gov:2672816; Copyright (c) International Union of Crystallography 2009; Country of input: International Atomic Energy Agency (IAEA); ISSN 0907-4449
- Country of Publication:
- Denmark
- Language:
- English
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