Neutron and light-scattering studies of DNA gyrase and its complex with DNA.
The solution structure of Escherichia coli DNA gyrase, an enzyme that catalyzes the ATP-dependent supercoiling of DNA, has been characterized by small-angle neutron scattering (SANS) and dynamic light-scattering (DLS). The enzyme and its complex with a 172 base-pair fragment of duplex DNA, in H2O or 2H2O solvent, were studied by contrast variation and the measurement of hydrodynamic parameters as a function of scattering angle. The complex was also measured in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP), a non-hydrolyzable ATP analog that is known to support limited supercoiling. The values of the radius of gyration, Rg = 67 A, from SANS and the hydrodynamic radius, Rh = 64 A, from DLS predict a larger than expected volume for the enzyme, supporting the notion of channels or cavities within the molecule. In addition, several classes of models were rejected based on SANS data obtained in 2H2O at larger scattering angles. The best fit to both the SANS and DLS data is obtained for oblate, inhomogeneous particles approximately 175 A wide and 52 A thick. Such particles provide a large surface area for DNA interaction. Both Rg and Rh values change very little upon addition of DNA, suggesting that DNA binds in a manner that does not significantly change the shape of the protein. No appreciable change in structure is found with the addition of ADPNP. However, the higher-angle SANS data indicate a slight rearrangement of the enzyme in the presence of nucleotide.
Krueger
S
S
National Institute of Standards and Technology, Gaithersburg, MD 20899.
Zaccai
G
G
Wlodawer
A
A
Langowski
J
J
O'Dea
M
M
Maxwell
A
A
Gellert
M
M
D000818:Animals; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008027:Light; D008433:Mathematics; D008961:Models, Structural; D008962:Models, Theoretical; D009502:Neutrons; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D012542:Scattering, Radiation; D012617:Sea Urchins
D004275:DNA, Ribosomal; D012341:RNA, Ribosomal, 5S; D004250:DNA Topoisomerases, Type II