Membrane Assembly of the Bacteriophage Pf3 Major Coat Protein

摘要

The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrnae of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly #alpha#-helical membrane-boudn protein traverses the membrane once, leaving hte C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrnae-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacety)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to teh surface of the membrane. A nearly identical result was seen previously for the membrnae-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an “L-shaped”membrane-boudn model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggests a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.