Characterization of two mutant lactose repressor proteins containing single tryptophans

Area of Science:

• Molecular Biology
• Protein Engineering
• Biochemistry

Background:

• Lactose repressor protein (LacI) regulates gene expression.
• Previous studies used amber suppression mutants to investigate LacI function.
• Site-specific mutagenesis offers an alternative approach to generate LacI mutants.

Purpose of the Study:

• To generate and characterize novel lactose repressor mutants using site-specific mutagenesis.
• To compare the binding properties of these new mutants with previously reported suppression mutants.
• To elucidate the roles of specific tryptophan residues (Trp201, Trp220) in repressor function.

Main Methods:

• Site-specific mutagenesis to introduce tryptophan substitutions (W201Y, W220Y).
• Protein expression and purification from plasmids.
• DNA binding assays (operator dissociation rate).
• Inducer binding studies.
• Spectroscopic analyses (fluorescence, UV-Vis, CD) and quenching experiments.

Main Results:

• Mutant plasmids produced stable, easily isolable proteins.
• W201Y and W220Y showed significantly reduced operator binding affinity compared to wild-type.
• Inducer binding affinities varied, with W220Y showing lower affinity than A220.
• Spectroscopic data indicated Trp201 is buried and Trp220 is exposed, with inducer binding affecting Trp220.
• Melibiose binding revealed distinct spectral features potentially involving Trp220.

Conclusions:

• Site-specific mutagenesis provides a robust method for generating functional lactose repressor mutants.
• Trp201 and Trp220 play distinct roles in DNA binding and inducer interaction.
• Trp220 may be involved in direct contact with disaccharide inducers.