Limited proteolytic digestion of lac repressor by trypsin. Chemical nature of the resulting trypsin-resistant core.
Tryptic digestion of Escherichia coli lac repressor under nondenaturing conditions readily removes 59 amino acids from the NH2-terminal end of the polypeptide chain. Longer digestion removes an additional 20 or more amino acids from the COOH terminus, leaving a highly trypsin-resistanct core molecule. The lac repressor tetrameric structure and inducer-binding activity are retained by the tryptic core. Operator-binding activity, however, is lost as the NH2-terminal end is degraded. Many or all of the possible trypsin cleavage sites in the NH2-terminal region are available to attack by the enzyme, indicating that this part of the polypeptide chain is exposed to the environment. Lac repressor, missing the NH2-terminal end, renatures efficiently from random-coil solvent to tetramers with full inducer-binding activity, indicating that the NH2-terminal region is not necessary for the appropriate three-dimensional folding of the polypeptide chains. Core which lacks both the NH2 and COOH termini renatures to tetramers with low efficiency.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004794:Enzyme Repression; D004926:Escherichia coli / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism*; D009154:Mutation; D009876:Operon; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013045:Species Specificity; D013862:Thiogalactosides / Q000378:metabolism; D014357:Trypsin
D001426:Bacterial Proteins; D010446:Peptide Fragments; D013862:Thiogalactosides; D014357:Trypsin; D007785:Lactose