J. Biochem, 1982, Vol. 91, No. 4 1213-1221
© 1982 Japanese Biochemical Society
research-article |
Structure of the 
tof Repressor Protein in Solution. Heat Stability and Its Relation to Binding Ability to DNA
*Institute for Protein Research, Osaka University Suita, Osaka 565
**Kyushu University Fukuoka 812
The 
tof repressor protein was purified from E. coli cells retaining dv plasmids by applying DNA-cellulose chromatography. 3H-labeled dv and imm21dv DNA, carrying and lacking operators, respectively, were prepared and the binding activity of the tof protein to the DNA was examined. Non-specific binding to imm21dv DNA is completely lost at 30°C, whereas specific binding to the DNA carrying the operators is retained even above 40°C.
The conformation of the tof protein was analysed by means of circular di-chroism and 1H-NMR spectra. The change in the molar ellipticity at 222 nm vs. temperature in CD spectra indicated a transition between two states with Tm at 42°C. The 360 MHz 1H-NMR spectra revealed the presence at 20°C of another change in local conformation or interaction which was not detected by the CD spectra. 1H-NMR also indicated the coexistence of thermal transitions with exchange rates faster and slower than the NMR time scale at about 50°C, which is explained by the presence of domain structures. The NMR titration curve of the His residue gave a normal pK value showing its location on the surface of the protein. These con-formational behaviors are well correlated to the specific and non-specific DNA binding activity of the tof protein. The assignments of 1H resonance signals to some specific residues, including His 35 and Tyr 26, were established. It will be useful to determine the tof–-DNA interaction.