Infrared attenuated total reflection (IR-ATR) spectroscopy and capacitance minimization (CM) were used to study the secondary structure, orientation, and accumulation of dynorphin A-(1-13)-tridecapeptide (dynorphin1-13) molecules on the surface of planar membranes prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. The peptide assumed a helical structure oriented perpendicularly on the membrane surface. Binding from aqueous solutions containing 10 mM KCl saturated reversibly at about a bilayer area of 110 nm2 per peptide molecule, an apparent dissociation constant of 11 microM, and rate constants of 2 X 10(2) s-1 (adsorption) and 2 X 10(-3) s-1 (desorption). The results complement those obtained by vesicle-mediated hydrophobic labeling [Gysin, B., & Schwyzer, R. (1983) Arch. Biochem. Biophys. 225, 467-474]. They indicate that the behavior of this amphiphilic peptide in contact with neutral lipid membranes may be quite different from that in molecularly disperse or micellar solutions of detergents or lysolecithins and that, in the case of dynorphin1-13, primary amphiphilicity overrules secondary amphiphilicity.

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Bombesin, in contact with flat phospholipid bilayer membranes, was shown to adopt a membrane structure similar to that of substance P, dynorphin-(1-13)-tridecapeptide, and adrenocorticotropin-(1-24)-tetracosapeptide. The C-terminal message segment, comprising 8-10 amino acid residues, is inserted into a relatively hydrophobic membrane compartment as an alpha-helical domain oriented perpendicularly on the membrane surface. The N-terminal, hydrophilic tetrapeptide segment remains in the aqueous compartment as a random coil. This was shown with IR and IR attenuated total reflection spectroscopy. Equilibrium thermodynamic estimations confirmed the observed membrane structure with respect to helix length, strength of hydrophobic membrane association, and orientation (caused by favorably oriented molecular amphiphilic and helix electric dipole moments). The membrane structure may explain why Trp-8 and His-12 are essential for biologic activity. Neuromedin B is predicted to be able to adopt a membrane structure similar to that of bombesin. However, gastrin-releasing peptide and neuromedin C are predicted not to behave in the same manner. The molecular mechanism of receptor subtype selection by bombesin-like peptides may prove to be similar to that observed earlier for opioid peptides and the neurokinins.

IR data of the neuropeptide substance P (1) and its synthetic segments des-(Arg'-Gin')-substance P (6), des-(Arg'-Pro4)-substance P (4), des-(Arg'-Lys3)-substance P (3), and des-Arg'-substance P (2) indicate predominant p-structures in the solid state and a-helical structures in CF,CH20H (amide I band shape analysis). In MeOH, the spectra of 1 suggest a partly helical structure. On membranes prepared from 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine, a C-terminal a-helix consisting of 8 or 9 peptide bonds appears to be induced (IR attenuated total reflection studies). Its perpendicular orientation on the membrane is suggested by the dichroic ratios of the amide-1 and -11 bands. This study is consistent with our CD experiments and lends support to the membrane structure of 1 predicted from the estimated amphiphilic moment, hydrophobic-association constant, and helix length.

Preferred conformation, orientation, and accumulation of substance P on a neutral hydrophilic-hydrophobic interface was estimated and extrapolated to interactions with neutral and anionic lipid bilayer membranes according to our general procedure. Nine residues at the C-terminus were predicted to he transferred to the hydrophobic phase as an a-helical domain, oriented quite perpendicularly on the membrane surface. The N-terminal residues remained in the aqueous phase with their charges exposed to H,O. The molecular amphiphilic moment vector was strong (338 arbitrary units) and pointed its hydrophilic end towards the N-terminus, only 15' away from the helix axis. The molecular electric dipole moment vector was also strong (124 debye) and pointed its positive end towards the N-terminus, only 9" away from the helix axis. Thus, it reinforced the effect of the amphiphilic moment of a peptide intruding into the membrane dipole layer. The estimated dissociation constant for the equilibrium between membrane-bound and free substance P was Kd = 46 mM for neutral membranes, and Kd % 0.43 mM for anionic membranes with a Gouy-Chapman surface potential of -40 mV. Thus, substance P behaved similarly to dynorphin A and adrenocorticotropin peptides which insert their N-terminal message segments as perpendicularly oriented helical domains into membranes, whereas their C-terminal address segments remain in the aqueous phase as random coils. Substance Pis the first instance of a neuropeptide which is expected to insert a C-terminal message into lipid membranes.