Site-directed mutants of the cAMP receptor protein-DNA binding of five mutant proteins

Oligonucleotide-directed mutagenesis was employed to generatemutants of the cAMP receptor protein (CRP) of Escherichia coli.The mutant proteins were purified to homogeneity and testedfor stability and DNA binding. It is shown that mutations atthe position of Arg¹⁸⁰ abolish specific DNA binding, whereasthose at the position Arg¹⁸⁵ have very little effect. Both positionshave previously been implicated as crucial for the specificinteraction between CRP and DNA. The Ser¹²⁸ Ala mutant showsa slight reduction in DNA binding affinity relative to wild-type.All mutants investigated show similar stability profiles towild-type CRP with respect to thermolysin proteolysis as a functionof temperature.