DOMAIN-STRUCTURE OF ESCHERICHIA-COLI DNA GYRASE AS REVEALED BY DIFFERENTIAL SCANNING CALORIMETRY

The domain structure of DNA gyrase from Escherichia coli has been exam ined using differential scanning microcalorimetry. The intact enzyme ( an A(2)B(2) tetramer) shows at least four transitions with apparent T- m's at 44.8, 53.3, 58.6, and 60.7 degrees C. Comparison with the therm al stabilities of the two separate subunits and genetically-engineered protein fragments has been used to assign these transitions to indivi dual domains within the intact gyrase proteins. The thermal unfolding of DNA gyrase and all individual fragments are irreversible under the conditions of the calorimetric experiment. Further evidence for the as signment of transitions to particular domains has been obtained by stu dying the effects of tight-binding ligands such as novobiocin on the t hermal stabilities of the various protein fragments.