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

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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 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 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.

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