Initial steps in protein membrane insertion. Bacteriophage M13 procoat protein binds to the membrane surface by...

Area of Science:

• Molecular Biology
• Biochemistry
• Membrane Biology

Background:

• Bacteriophage M13 procoat protein is synthesized on free polysomes.
• It is a precursor protein that inserts into the inner membrane of Escherichia coli.
• Initial interaction with the cytoplasmic face of the inner membrane is a key step in its pathway.

Purpose of the Study:

• To investigate the regions of M13 procoat protein responsible for binding to the E. coli inner membrane.
• To elucidate the mechanism of initial membrane interaction for M13 procoat protein.

Main Methods:

• Oligonucleotide-directed mutagenesis was employed to create M13 procoat protein mutants.
• Mutant proteins were analyzed for their ability to interact with the E. coli inner membrane.
• In vitro experiments using artificial liposomes were conducted to test binding interactions.

Main Results:

• Positively charged amino acids at both ends of the M13 procoat protein were found to be essential for membrane binding.
• Mutations replacing positive charges with negative charges led to precursor accumulation in the cytoplasm.
• Wild-type procoat protein bound to artificial liposomes, while mutants with negative charges at both ends did not.

Conclusions:

• Newly synthesized M13 procoat protein utilizes electrostatic interactions for binding to the negatively charged E. coli inner membrane.
• The positively charged amino acids at the protein termini play a direct role in this electrostatic membrane association.