Structure of the λ tof Repressor Protein in Solution. Heat Stability and Its Relation to Binding Ability to DNA

Abstract

The λ tof repressor protein was purified from E. coli cells retaining λdv plasmids by applying DNA-cellulose chromatography. ³H-labeled λdv and λimm²¹dv DNA, carrying and lacking λ operators, respectively, were prepared and the binding activity of the λ tof protein to the DNA was examined. Non-specific binding to λimm²¹dv 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 λ tof protein was analysed by means of circular dichroism 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 T_m 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 λ tof protein. These conformational behaviors are well correlated to the specific and non-specific DNA binding activity of the λ 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.