Mutation in hinge region of lactose repressor protein alters physical and functional properties.
A mutant of the Escherichia coli lactose repressor (BG124) in which serine at position 77 is replaced by leucine has been examined by physical methods. Consistent with the phenotypic character of this i-d mutant, BG124 protein did not bind lactose operator specifically, but did bind to DNA nonspecifically. Titration with inducer monitoring tryptophan fluorescence changes yielded a biphasic saturation curve, and Scatchard and Hill plots of the fluorescence and equilibrium dialysis data demonstrated heterogeneity of inducer binding sites. Although ultraviolet difference spectra and potassium iodide quenching of fluorescence indicated that BG124 repressor has structural distinctions from wild-type protein, circular dichroism spectra and acrylamide quenching of fluorescence for the two proteins were quite similar. A significantly greater increase of 1-anilino-8-naphthalenesulfonate fluorescence was observed in the presence of mutant versus wild-type repressor. Unlike wild-type behavior, changes in both 1-anilino-8-naphthalenesulfonate fluorescence intensity and maximum emission wavelength in response to inducer were found for the BG124 protein. These results are consistent with conformational alterations in the interface between NH2-terminal and core domains of this mutant repressor. The single amino acid alteration in the hinge between the core and NH2 terminus yields conformational effects which influence physical and functional properties associated with both domains.
Chou
W Y
WY
Department of Biochemistry, Rice University, Houston, Texas 77251.
Matthews
K S
KS
D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D009154:Mutation; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D007544:Isopropyl Thiogalactoside