Membrane-anchoring interactions of M13 major coat protein

The response to hydrophobic mismatch of membrane-bound M13 major coat prote in is measured using site-directed fluorescence and ESR spectroscopy. For t his purpose, we investigate the membrane-anchoring interactions of M13 coat protein in model systems consisting of phosphatidylcholine bilayers that v ary in hydrophobic thickness. Mutant coat proteins are prepared with an AED ANS-labeled single cysteine residue in the hinge region of the protein or a t the C-terminal side of the transmembrane helix. In addition, the fluoresc ence of the tryptophan residue is studied as a monitor for the N-terminal s ide of the transmembrane helix. The fluorescence results show that the hing e region and C-terminal side of the transmembrane helix hardly respond to h ydrophobic mismatch. In contrast, the N-terminal side of the helical transm embrane domain shifts to a more apolar environment, when the hydrophobic th ickness is increased. The apparent strong membrane-anchoring interactions o f the C-terminus are confirmed using a mutant that contains a longer transm embrane domain. As a result of this mutation, the tryptophan residue at the N-terminal side of the helical domain clearly shifts to a more polar envir onment, whereas the labeled position 46 at the C-terminal side is not affec ted. The phenylalanines in the C-terminal part of the protein play an impor tant role in these apparent strong anchoring interactions. This is demonstr ated with a mutant in which both phenylalanines are replaced by alanine res idues. The phenylalanine residues in the C-terminus affect the location in the membrane of the entire transmembrane domain of the protein.