Unfolding of lac repressor and its proteolytic fragment by urea: headpieces stabilize the core within lac repressor.
Circular dichroism measurements were used to compared the urea-induced unfolding transition of the lac repressor with those of its separated tryptic fragments, the tetrameric core, and the N-terminal headpiece. The presence of the headpieces covalently linked to the core in the intact repressor leads to a stabilization against urea denaturation as compared to that for the isolated core. This results in a shift of the midpoint of the transition by about 0.5 M urea. When the inducer isopropyl beta-D-thiogalactoside is bound, the core is stabilized more than the entire repressor. The isolated headpiece is considerably more stable against urea denaturation than the tryptic core or the lac repressor. The reversible denaturation process of the headpiece was quantitatively analyzed, and the free energy of unfolding in the absence of urea was found to be 2.4 or 2.9 kcal/mol, depending on the method of calculation used. Comparison between the circular dichroism spectra of the lac repressor, the tryptic core of the lac repressor, and the headpiece supply further evidence that there are no major conformational differences between the structural domains (core and headpieces) before and after proteolytic cleavage of the lac repressor. These results are discussed with respect to the contacts between the different domains of the protein. It is concluded that relatively weak interdomain contacts are probably responsible for the stabilization of the core by the covalently linked headpieces and that these contacts might be weakened upon binding of the inducer.
D002942:Circular Dichroism; D004355:Drug Stability; D004926:Escherichia coli / Q000032:analysis; D010446:Peptide Fragments; D011487:Protein Conformation; D011489:Protein Denaturation; D012097:Repressor Proteins; D013816:Thermodynamics; D014157:Transcription Factors; D014508:Urea
D010446:Peptide Fragments; D012097:Repressor Proteins; D014157:Transcription Factors; D014508:Urea