Papain does not cleave operator-bound lambda repressor: structural characterization of the carboxy terminal domain...

Area of Science:

• Molecular Biology
• Protein Structure
• Biochemistry

Background:

• The bacteriophage lambda cI repressor is crucial for viral gene regulation.
• Understanding its structural domains is key to deciphering its function.
• Previous studies suggested a 'hinge' region, but its structure was unclear.

Purpose of the Study:

• To investigate the secondary structure and dimerization properties of carboxy-terminal fragments of the lambda cI repressor.
• To determine the structural role of the 93-131 region.
• To compare the behavior of wild-type and mutant repressor fragments.

Main Methods:

• Circular dichroism (CD) spectroscopy to analyze secondary structure.
• Fourier-transformed infrared (FTIR) spectroscopy for structural confirmation.
• High-performance liquid chromatography (HPLC) to assess protein dimerization.
• Papain cleavage assays to study protein stability and processing.
• Tryptophan fluorescence spectroscopy to monitor protein denaturation.

Main Results:

• Carboxy-terminal fragments (93-236, 112-236, 132-236) are rich in beta-strands and loops, with increasing helix content in larger fragments.
• The 93-131 region is structured, not a flexible hinge.
• Fragment 132-236 lacks detectable alpha-helix, containing only beta-sheets and turns.
• Dimerization properties of wild-type and SN228 mutant fragments were similar.
• Papain cleavage rates differed between wild-type and SN228 mutants.
• Cysteine residues in fragment 132-236 are buried and unreactive.
• Denaturation of fragment 132-236 revealed two transitions.

Conclusions:

• The carboxy-terminal domain of the lambda cI repressor possesses a defined structure.
• The 93-131 region contributes to the structural integrity of the repressor.
• Dimerization and proteolytic cleavage are influenced by specific mutations within the repressor.
• The buried nature of cysteine residues suggests a compact structure in the carboxy-terminal domain.