Thermal denaturation of engineered tet repressor proteins and their complexes with tet operator and tetracycline studied by temperature gradient gel electrophoresis.

The effects of Trp to Phe exchanges in the Tet repressor on the thermal stability of the proteins and their complexes with operator DNA and inducer have been studied by temperature gradient polyacrylamide gel electrophoresis. The denaturation temperatures obtained by this method are compared with the results from temperature-dependent fluorescence and binding activities of the proteins. It is established that exchanging the interior Trp75 to Phe reduces the thermal stability of the Tet repressor by 8 degrees C while exchanging the exterior Trp43 to Phe has no effect on the stability of the protein. Binding of the inducer tetracycline increases the thermal stability of wild-type and Trp43 to Phe mutant Tet repressors by 5 degrees C, while the ones with the Trp75 to Phe mutation are stabilized by 10 degrees C. The stabilizing effect of operator binding is 20 degrees C in the Trp75 to Phe mutant and only 9 degrees C in the ones with the Trp43 to Phe exchange. In addition to the denaturation temperatures, the gel mobility shifts observed in temperature gradient gel electrophoresis reveal also information about the intermediates of the denaturation reaction. The free proteins and their complexes with the inducer tetracycline exhibit monophasic transitions upon denaturation. The operator complexes of wild-type and Trp75 to Phe mutant repressors denature in more complex reactions. At low temperature they exhibit a stoichiometry of two repressor dimers per tandem tet operator DNA. Upon elevating the temperature they form complexes with only one repressor dimer per DNA fragment. When the temperature is further increased the double-stranded DNA begins to melt from one end resulting in a complex with partially single-stranded DNA which exists only in a narrow temperature range. Finally, the denatured protein and single-stranded DNA are formed at high temperature. The associated mobility shifts are analyzed by changing the ionic strength and characterizing multiphasic melting of a pure DNA fragment by temperature gradient gel electrophoresis.