Thermodynamic computational approach to capture molecular recognition in the binding of different inhibitors to the DNA gyrase B subunit from Escherichia coli

The 24 kDa fragment of DNA gyrase B from Staphylococcus aureus was expressed in Escherichia coli and purified for crystallization. Crystals of the wild-type protein grew in the presence of cyclothialidine but proved difficult to reproduce. In order to improve the crystallization, the flexible regions of the protein were deleted by mutagenesis. The mutant proteins were analyzed by differential scanning calorimetry and the most stable mutants produced crystals. It was possible to reproducibly grow in the microbatch system single well defined crystals which belonged to the space group C2 and diffracted isotropically to approximately 2 Å resolution.

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Antagonism of the B subunit of DNA gyrase eliminates plasmids pBR322 and pMG110 from Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.152.1.338-344.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 338-344

Author(s):

J S Wolfson ◽

D C Hooper ◽

M N Swartz ◽

G L McHugh

Keyword(s):

Escherichia Coli ◽

Dna Gyrase ◽

B Subunit ◽

Plasmid Loss ◽

Escherichia Coli Cells ◽

Bacterial Mutant ◽

Bacterial Enzyme ◽

Kinetics Of ◽

Plasmid Elimination ◽

Native Plasmid

The constructed plasmid pBR322 and the native plasmid pMG110 were eliminated (cured) from growing Escherichia coli cells by the antagonism of the B subunit of the bacterial enzyme DNA gyrase. The antagonism may be by the growth of cells (i) at semipermissive temperatures in a bacterial mutant containing a thermolabile gyrase B subunit or (ii) at semipermissive concentrations of coumermycin A1, an antibiotic that specifically inhibits the B subunit of DNA gyrase. The kinetics of plasmid elimination indicate that plasmid loss occurs too rapidly to be explained solely by the faster growth of that plasmid-free bacteria and, therefore, represents interference with plasmid maintenance.

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Crystal engineering: a case study using the 24 kDa fragment of the DNA gyrase B subunit from Escherichia coli

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444999008136 ◽

1999 ◽

Vol 55 (9) ◽

pp. 1623-1625 ◽

Cited By ~ 20

Author(s):

Allan D'Arcy ◽

Martine Stihle ◽

Dirk Kostrewa ◽

Glenn Dale

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Crystal Engineering ◽

Dna Gyrase ◽

Site Directed Mutagenesis ◽

Crystal Quality ◽

Single Amino Acid ◽

Wild Type ◽

B Subunit

Site-directed mutagenesis was used to determine the efficacy of changing surface residues to improve crystal quality. Nine mutants of the 24 kDa fragment of the Escherichia coli DNA gyrase B subunit were produced, changing residues on the protein's surface. The mutations changed either the charge or the polarity of the wild-type amino acid. It was found that single amino-acid changes on the surface could have a dramatic effect on the crystallization properties of the protein and generally resulted in an improvement in the number of crystal-screen hits as well as an improvement in crystal quality. It is concluded that crystal engineering is a valuable tool for protein crystallography.

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