dynorphins and 1-palmitoyl-2-oleoylphosphatidylcholine

The membrane interaction properties of two single-residue variants, R6W and L5S, of the 17-amino acid neuropeptide dynorphin A (DynA) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Corresponding gene mutations have recently been discovered in humans and causatively linked to a neurodegenerative disorder. The peptides were investigated in buffer and in isotropic solutions of q = 0.3 bicelles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or DMPC (0.8) and 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DMPG) (0.2). The CD results and the NMR secondary chemical shifts show that R6W-DynA has a small α-helical fraction in buffer, which increases in the presence of bicelles, while L5S-DynA is mainly unstructured under all conditions studied here. R6W-DynA has an almost complete association with zwitterionic bicelles (∼90%, as probed by NMR diffusion experiments), similar to the behavior of wtDynA, while L5S-DynA has a weaker association (∼50%). For all peptides, the level of bicelle association is increased in negatively charged bicelles. The L5A-DynA peptide adopts a very shallow position in the headgroup region of the bicelle bilayer, as studied by paramagnetic spin relaxation enhancement experiments using paramagnetic probes. Similarly, the results show that R6W-DynA is more deeply buried in the bilayer, with only the C-terminal residues exposed to solvent, again more similar to the case of wild-type DynA. We suggest that the results presented here may explain the differences in cell toxicity of these disease-related neuropeptide variants.

Topics: Cell Membrane; Circular Dichroism; Diffusion; Dimyristoylphosphatidylcholine; Dynorphins; Humans; Lipid Bilayers; Magnetic Resonance Spectroscopy; Micelles; Mutation; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Phospholipid Ethers; Protein Conformation; Solvents; Thermodynamics; Water

Dynorphins, endogeneous opioid peptides, function as ligands to the opioid kappa receptors and induce non-opioid excitotoxic effects. Here we show that big dynorphin and dynorphin A, but not dynorphin B, cause leakage effects in large unilamellar phospholipid vesicles (LUVs). The effects parallel the previously studied potency of dynorphins to translocate through biological membranes. Calcein leakage caused by dynorphin A from LUVs with varying POPG/POPC molar ratios was promoted by higher phospholipid headgroup charges, suggesting that electrostatic interactions are important for the effects. A possibility that dynorphins generate non-opioid excitatory effects by inducing perturbations in the lipid bilayer of the plasma membrane is discussed.

Topics: Amino Acid Sequence; Dynorphins; Endorphins; Fluoresceins; Liposomes; Membranes, Artificial; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylglycerols; Phospholipids

Equilibrium thermodynamic and kinetic estimations were used to confirm the rather unusual conformation, orientation, and accumulation of dynorphin A-(1-13)-tridecapeptide (dynorphin1-13) on the surface of neutral lipid membranes, as observed by Erne et al. [Erne, D., Sargent, D. F., & Schwyzer, R. (1985) Biochemistry 24, 4261-4263]. I started from the premise that the most stable conformation of molecularly disperse peptides in contact with the hydrophobic phase of a membrane is helical [Henderson, R. (1979) Soc. Gen. Physiol. Ser. 33, 3-15]. Calculation of the Gibbs free energy difference for the transfer of increasing numbers m of N-terminal residues of dynorphin1-13 from their random-coil conformation in water to their alpha-helical conformation in a hydrophobic phase, with the values provided by Von Heijne and Blomberg [Von Heijne, G., & Blomberg, C. (1979) Eur. J. Biochem. 97, 175-181], showed an energy minimum at m = 9 that corresponded to the observed apparent association constant of 9 X 10(4) L/mol. This confirmed our experimental observations. The orientation of dynorphin1-13 in the interphase was estimated by calculation of the molecular amphiphilic moment A. This force vector was defined in analogy to the "helical" and "structural" hydrophobic moments of Eisenberg et al. [Eisenberg, D., Weiss, R. M., & Terwilliger, T. C. (1982) Nature (London) 299, 371-374]. It takes into account the segregation of hydrophobic and hydrophilic residues with respect to the center of the alpha-helix. A peptide located in a hydrophobic-hydrophilic gradient experiences a torque that tends to orient A in a direction perpendicular to the surfaces of equal hydrophobicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Dynorphins; Liposomes; Mathematics; Peptide Fragments; Phosphatidylcholines; Protein Conformation; Thermodynamics