Polarized infrared absorption of Na+/K+-ATPase studied by attenuated total reflection spectroscopy.

Na+/K+-ATPase can be isolated from the outer medulla of mammalian kidney in the form of flat membrane fragments containing the enzyme in a density of 10(3)-10(4) protein molecules per microm2 (Deguchi et al. (1977) J. Cell. Biol. 75, 619-634). In this paper we show that these membrane fragments can be bound to a germanium plate coated with a phospholipid bilayer. With this system infrared spectroscopic studies of the enzyme have been carried out using the technique of attenuated total reflection (ATR). At a coverage of the lipid surface corresponding to 30-40% of a monolayer of membrane fragments, characteristic infrared bands of the protein such as the amide I and II bands can be resolved. About 24% of the NH-groups of the peptide backbone are found to be resistant to proton/deuterium exchange within a time period of several days. Evidence for orientation of the protein with respect to the supporting lipid layer is obtained from experiments with polarized light, the largest polarization effects being associated with the -COO- band at 1400 cm-1. Experiments with aqueous media of different ionic composition indicate that the average orientation of transition moments changes when K+ in the medium is replaced by Tris+ or Na+.

Structure-activity relationship in vinculin: an IR/attenuated total reflection spectroscopic and film balance study.

Surfacing and membrane-penetrating ability of vinculin and bovine serum albumin have been studied on a macroscopic level by means of a Langmuir film balance and on a molecular level by means of infrared attenuated total reflection spectroscopy. It is suggested that the driving force of the nonspontaneous process of membrane penetration by native vinculin is the spontaneous formation of rigid vinculin monolayers in the membrane. Lateral adhesion of vinculin molecules results from the formation of intermolecular pleated-sheet structures. Vinculin surface activity was found to result from an alpha-helical segment oriented approximately perpendicular to plane of the membrane. There is a conformational equilibrium between this helix and random structure. High ionic strength (110 mM) favors helix formation that leads to the greater than 100-fold enhancement of surfacing velocity relative to the velocity observed at a lower ionic strength (10 mM). Vinculin has a second helical segment oriented parallel to the plane of the membrane that is in a conformational equilibrium with the pleated-sheet structure.

Pore formation in lipid membranes by alamethicin.

The conformation of the linear peptide antibiotic alamethicin in dipalmitoyl phosphatidylcholine multilayers was investigated in the absence of an electric field by means of infrared attenuated total reflection spectroscopy. Alamethicin was found to be incorporated into the lipid membrane not only in the dry state but also in an aqueous environment. Its molecular conformation, however, changed from a helix when dry to an extended chain when aqueous. The extended chain aggregated to di- and multimers spanning the lipid bilayer. The equilibrium concentration of alamethicin in the surrounding water was 90 nM, which is in the range of concentrations used in black film experiments. The corresponding molar ratio of lipid to peptide was 80:1. Concerning the molecular mechanism of electric field-induced pore formation, one has to conclude that the dipole model proposed by several authors is very unlikely because it is based on the assumption that the major part of alamethicin is adsorbed on the membrane surface, from which small amounts flip into the membrane under the influence of an electric field. An alternative mechanism is proposed, based on a field-induced conformational change of the peptide from the extended state to a helix. This transition is favored by the resulting dipole moment of the alamethicin helix.