Neutron diffraction studies of amphipathic helices in phospholipid bilayers.

The structural feature which is thought to facilitate the interaction of many peptides with phospholipid bilayers is the ability to fold into an amphipathic helix. In most cases the exact location and orientation of this helix with respect to the membrane is not known, and may vary with factors such as pH and phospholipid content of the bilayer. The growing interest in this area is stimulated by indications that similar interactions can contribute to the binding of certain hormones to their cell-surface receptors. We have been using the techniques of neutron diffraction from stacked phospholipid bilayers in an attempt to investigate this phenomenon with a number of membrane-active peptides. Here we report some of our findings with three of these: the bee venom melittin; the hormone calcitonin; and a synthetic peptide representing the ion channel fragment of influenza A M2 protein.

Neutron diffraction reveals the site of amantadine blockade in the influenza A M2 ion channel.

The influenza A M2 protein forms proton channels which are blocked by the anti-influenza drug amantadine. Using the technique of neutron diffraction with both deuterium-labeled amantadine and influenza A M2 peptides, this study has directly located the position of interaction between the drug and the transmembrane domain of M2. Amantadine is found 0.5 nm from the center of the bilayer in an area between Val 27 and Ser 31, a location consistent with the formation of a steric block within the ion channel. Similar experiments with amantadine and an amantadine-resistant mutant peptide showed no such interaction.

The location of amantadine hydrochloride and free base within phospholipid multilayers: a neutron and X-ray diffraction study.

Concomitant neutron and X-ray studies were undertaken in order to locate accurately the anti-influenza and Parkinson's disease drug amantadine in multilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine. The X-ray data were phased using the swelling series method and the neutron data were phased using D2O/H2O exchange and a variation of the isomorphous replacement technique. The sets of data complement each other and reveal two populations of amantadine within the bilayer. One site is close to the bilayer surface, the other is much deeper. The majority of the amantadine occupies the surface site. The relative occupancy, but not the position, of the two locations appears to be dependent upon the initial protonation state of the drug. No evidence of bilayer perturbation was observed with either the protonated or the deprotonated forms of amantadine.

The secondary structure of influenza A M2 transmembrane domain. A circular dichroism study.

Using circular dichroism, this study investigated the secondary structure of the influenza A M2 transmembrane domain. When reconstituted into 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes, the M2 transmembrane domain was found to adopt a predominantly alpha-helical secondary structure which was unaffected by both temperature and the addition of 1-aminoadamantane hydrochloride. Reconstitution into 1,2-dioleoyl-sn-glycero-3-phosphoglycerol liposomes resulted in a marked decrease in helical content.

The transmembrane domain of influenza A M2 protein forms amantadine-sensitive proton channels in planar lipid bilayers.

In a direct test of the hypothesis that the M2 coat protein of influenza A can function as a proton translocator, we incorporated a synthetic peptide containing its putative transmembrane domain into voltage-clamped planar lipid bilayers. We observed single proton-selective ion channels with a conductance of approximately 10 pS at a pH of 2.3, consistent with the association of several monomers around a central water-filled pore. The channels were reversibly blocked by the anti-influenza drug amantadine. These experiments imply a central role for M2 protein in virus replication and assembly and may explain the mechanism of action of amantadine. Analogous proteins may have a similar function in other viruses, and these may be susceptible to similar antiviral agents.