Fluorescence resonance energy transfer shows a close helix-helix distance in the transmembrane M13 procoat protein.
During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helices 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 molecular mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helices might drive the actual translocation process. We investigated the intramolecular distance between the two helices 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 molecular modeling, show that the transmembrane helices are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for determining the topology and the folding of membrane proteins.
Eisenhawer
M
M
Institute of Physical Chemistry, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland.
Cattarinussi
S
S
Kuhn
A
A
Vogel
H
H
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000737:chemistry* / Q000235:genetics; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D036022:Capsid Proteins; D004735:Energy Transfer; D005453:Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D036022:Capsid Proteins; D011498:Protein Precursors; C025668:coat protein, Bacteriophage M13; D014443:Tyrosine; D014364:Tryptophan