Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 inmodel membranes

Gene 9 minor coat protein from bacteriophage M13 is known to be located in the inner membrane after phage infection of Escherichia coli. The way of in sertion of this small protein (32 amino acids) into membranes is still unkn own. Here we show that the protein is able to insert in monolayers. The lim iting surface pressure of 35 mN/m for 1,2-dioleoyl-sn-glycero-3-phosphochol ine and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol lipid systems indicates t hat this spontaneous insertion can also occur in vivo. By carboxyfluorescei n leakage experiments of vesicles it is demonstrated that protein monomers, or at least small aggregates, are more effective in releasing carboxyfluor escein than highly aggregated protein. The final orientation of the protein in the bilayer after insertion was addressed by proteinase K digestion, th ereby making use of the unique C-terminal location of the antigenic binding site. After insertion the C-terminus is still available for the enzymatic digestion, while the N-terminus is not. This leads to the overall conclusio n that the protein is able to insert spontaneously into membranes without t he need of any machinery or transmembrane gradient, with the positively cha rged C-terminus remaining on the outside. The orientation after insertion o f gene 9 protein is in agreement with the 'positive inside rule'. (C) 2001 Elsevier Science B.V. All rights reserved.