Papain does not cleave operator-bound lambda repressor: structural characterization of the carboxy terminal domain and the hinge.
The circular dichroism spectra of three different purified carboxy terminal fragments 93-236, 112-236 and 132-236 of the bacteriophage lambda cI repressor have been measured and compared with those of the intact repressor and the amino terminal fragment 1-92. All three carboxy terminal fragments contain mostly beta-strands and loops, a minor helix content increasing with the size of the fragment, showing that the 93-131 region previously called a hinge is structured. Fourier transformed infrared spectra also showed that fragment 93-236 contains alpha-helices, alpha-sheets and turns but fragment 132-236 contains no detectable alpha-helix, only beta-sheets and turns. Papain is known to cleave the lambda repressor, but it is shown here that it cannot cleave the operator-bound repressor dimer. For the 132-236 fragment, both the wt and the SN228 mutant previously shown to be dimerization defective in the intact, gave similar dimerization properties as investigated by HPLC at 2 to 100 microM protein concentration, with a KD of 13.2 microM and 19.1 microM respectively. The papain cleavage for wt and SN228 proceed at equal rates for the first cleavage at 92-93; however, the subsequent cleavages are faster for SN228. The three Cys residues in the 132-236 fragment were found to be unreactive upon incubation with DTNB, indicating the thiol sulfur atoms are buried in the repressor carboxy terminal domain. Denaturation of the 132-236 fragment studied by tryptophan fluorescence shows two transitions centered at 1.5 M and 4.5 M of urea.
Ghosh
K
K
Department of Biochemistry, Bose Institute, Calcutta, India.
Chattopadhyaya
R
R
D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D019281:Dimerization; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D009875:Operator Regions, Genetic / Q000235:genetics*; D010206:Papain / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D014508:Urea / Q000737:chemistry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D014508:Urea; D004247:DNA; D004228:Dithionitrobenzoic Acid; D010206:Papain