G117C MelB, a mutant melibiose permease with a changed conformational equilibrium

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Transport

Background:

• Melibiose permease is a crucial transporter protein.
• Understanding transporter mechanisms requires detailed kinetic analysis.
• Mutations can reveal insights into protein function and dynamics.

Purpose of the Study:

• To investigate the functional and kinetic consequences of a glycine to cysteine substitution (G117C) in the melibiose permease.
• To elucidate the impact of this mutation on transporter conformation and pre-steady-state kinetics.
• To propose a kinetic model for the mutant transporter and discuss its implications for the wild type.

Main Methods:

• Site-directed mutagenesis to create the G117C mutant.
• Transport assays to measure melibiose permease activity.
• Pre-steady-state kinetic measurements, including substrate concentration jumps and fluorescence changes.

Main Results:

• The G117C mutant retained transport activity comparable to the wild type.
• Pre-steady-state kinetic properties, such as transient charge displacements and fluorescence changes, were significantly reduced.
• These findings indicate altered initial conformation of the mutant transporter despite maintained translocation function.

Conclusions:

• The G117C mutation alters the dynamic equilibrium of the substrate-free transporter.
• A kinetic model involving a rapid dynamic equilibrium of the substrate-free transporter is proposed for the G117C mutant.
• The study provides insights into the transport mechanism of the wild type melibiose permease by comparison.