Structure of the lambda tof repressor protein in solution. Heat stability and its relation to binding ability to DNA.
The lambda tof repressor protein was purified from E. coli cells retaining lambda dv plasmids by applying DNA-cellulose chromatography. 3H-labeled lambda dv and lambda imm21dv DNA, carrying and lacking lambda operators, respectively, were prepared and the binding activity of the lambda tof protein to the DNA was examined. Non-specific binding to lambda imm21dv DNA is completely lost at 30 degrees C, whereas specific binding to the DNA carrying the operators is retained even above 40 degrees C. The conformation of the lambda 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 Tm at 42 degrees C. The 360 MHz 1H-NMR spectra revealed the presence at 20 degrees C of another change in local conformation of 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 degrees 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 conformational behaviors are well correlated to the specific and non-specific DNA binding activity of the lambda 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.
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000032:analysis*; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions; D013816:Thermodynamics; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D012996:Solutions; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA