TY - JOUR
T1 - Stability and DNA-binding properties of the ω regulator protein from the broad-host range Streptococcus pyogenes plasmid pSM19035
AU - Misselwitz, Rolf
AU - De La Hoz, Ana B.
AU - Ayora, Silvia
AU - Welfle, Karin
AU - Behlke, Joachim
AU - Murayama, Kazutaka
AU - Saenger, Wolfram
AU - Alonso, Juan C.
AU - Welfle, Heinz
N1 - Funding Information:
We thank Mrs. B. Kannen for technical assistance. A.B.d.l.H. and S.A. were recipients of fellowships of the Gobierno Vasco and Ministerio de Educación, respectively. This work was partially supported by Grants BMC2000-0548 from MCT-DGI and BIO4-CT98-0099 to J.C.A., by Fonds der Chemischen Industrie and EU-Grant BIO4-CT98-0106 to W.S., and by Deutsche Forschungsgemeinschaft to H.W. (GRK 80/2 Partial Project and We1745/5-1) and W.S. (Sa196/38-1).
PY - 2001/9/21
Y1 - 2001/9/21
N2 - At the transcriptional level, the pSM19035-encoded ω protein coordinates the expression of proteins required for control of copy number and maintenance of plasmids. Using circular dichroism, fluorescence spectroscopy, ultracentrifugation and an electrophoretic mobility shift assay, the wild-type ω protein and a variant with a C-terminal hexa-histidine tag (ω-H6) were characterized. The ω protein is mainly α-helical (42%), occurs as homodimer in solution, unfolds thermally with half transition temperatures, Tm, between ∼43 and ∼78°C depending on the ionic strength of the buffer, and binds PcopS-DNA with high affinity. The ω-H6 protein has a modified conformation with lower α-helix content (29%), lower thermal stability, and strongly reduced affinity to PcopS-DNA.
AB - At the transcriptional level, the pSM19035-encoded ω protein coordinates the expression of proteins required for control of copy number and maintenance of plasmids. Using circular dichroism, fluorescence spectroscopy, ultracentrifugation and an electrophoretic mobility shift assay, the wild-type ω protein and a variant with a C-terminal hexa-histidine tag (ω-H6) were characterized. The ω protein is mainly α-helical (42%), occurs as homodimer in solution, unfolds thermally with half transition temperatures, Tm, between ∼43 and ∼78°C depending on the ionic strength of the buffer, and binds PcopS-DNA with high affinity. The ω-H6 protein has a modified conformation with lower α-helix content (29%), lower thermal stability, and strongly reduced affinity to PcopS-DNA.
KW - Circular dichroism
KW - Fluorescence
KW - Plasmid copy number control
KW - Protein stability
KW - Protein-DNA interaction
KW - Thermal unfolding
KW - Transcriptional repressor
KW - Urea unfolding
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U2 - 10.1016/S0014-5793(01)02865-4
DO - 10.1016/S0014-5793(01)02865-4
M3 - Article
C2 - 11576543
AN - SCOPUS:0035929158
SN - 0014-5793
VL - 505
SP - 436
EP - 440
JO - FEBS Letters
JF - FEBS Letters
IS - 3
ER -