During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helixes corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other. Previous studies on the mol. mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helixes might drive the actual translocation process. We investigated the intramol. distance between the two helixes of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix. Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers. The results obtained from the FRET measurements, combined with mol. modeling, show that the transmembrane helixes are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for detg. the topol. and the folding of membrane proteins. [on SciFinder (R)]