TY - JOUR
T1 - Conformational transition of DnaA protein by ATP
T2 - Structural analysis of DnaA protein, the initiator of Escherichia coli chromosome replication
AU - Kubota, Toshio
AU - Katayama, Tsutomu
AU - Ito, Yuji
AU - Mizushima, Tohru
AU - Sekimizu, Kazuhisa
N1 - Funding Information:
The overproducer of DnaA protein was constructed in the laboratory of Dr. A. Kornberg by the second author, who was supported there in part by the International Human Frontier Science Program. We are grateful to Drs. E. Crooke, D.-S. Hwang, D. Bramhill, and N. E. Dixon for suggestions on overproduction and puri®cation of DnaA protein. We thank Dr. T. Ueda and M. Ohara for suggestions on analysis of CD data and for comments on the manuscript, respectively. This work was supported in part by Grants-in-Aid for Scien-ti®c Research from the Ministry of Education, Science, Sports, and Culture of Japan.
PY - 1997/3/6
Y1 - 1997/3/6
N2 - DnaA protein binds to the chromosomal origin (oriC) to initiate DNA replication. We developed an efficient system for purification of DnaA protein which will facilitate physicochemical analysis of the protein. The yield of DnaA protein was increased at least 6-fold compared to an available method being used, and over 22 mg of the protein were obtained from only 100 g of cells. DnaA protein purified by this procedure showed an indistinguishable affinity for ATP, and activity for in vitro replication of oriC plasmid. The process of denaturation of DnaA protein, which was blocked by ATP, was monitored by intrinsic fluorescence and circular dichroism. Analysis of circular dichroism revealed that DnaA protein is rich in α-helices, and that ATP-binding leads to a significant transition of protein conformation in that the content of α-helices is decreased. This is the first evidence indicating that ATP-binding profoundly affects conformation of DnaA protein.
AB - DnaA protein binds to the chromosomal origin (oriC) to initiate DNA replication. We developed an efficient system for purification of DnaA protein which will facilitate physicochemical analysis of the protein. The yield of DnaA protein was increased at least 6-fold compared to an available method being used, and over 22 mg of the protein were obtained from only 100 g of cells. DnaA protein purified by this procedure showed an indistinguishable affinity for ATP, and activity for in vitro replication of oriC plasmid. The process of denaturation of DnaA protein, which was blocked by ATP, was monitored by intrinsic fluorescence and circular dichroism. Analysis of circular dichroism revealed that DnaA protein is rich in α-helices, and that ATP-binding leads to a significant transition of protein conformation in that the content of α-helices is decreased. This is the first evidence indicating that ATP-binding profoundly affects conformation of DnaA protein.
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U2 - 10.1006/bbrc.1997.6244
DO - 10.1006/bbrc.1997.6244
M3 - Article
C2 - 9125116
AN - SCOPUS:0031555893
SN - 0006-291X
VL - 232
SP - 130
EP - 135
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -