2-arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor.

Two types of endogenous cannabinoid-receptor agonists have been identified thus far. They are the ethanolamides of polyunsaturated fatty acids--arachidonoyl ethanolamide (anandamide) is the best known compound in the amide series--and 2-arachidonoyl glycerol, the only known endocannabinoid in the ester series. We report now an example of a third, ether-type endocannabinoid, 2-arachidonyl glyceryl ether (noladin ether), isolated from porcine brain. The structure of noladin ether was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by comparison with a synthetic sample. It binds to the CB(1) cannabinoid receptor (K(i) = 21.2 +/- 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB(2) receptor (K(i) > 3 microM).

Solution structures of a highly insecticidal recombinant scorpion alpha-toxin and a mutant with increased activity.

The solution structure of a recombinant active alpha-neurotoxin from Leiurus quinquestriatus hebraeus, Lqh(alpha)IT, was determined by proton two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). This toxin is the most insecticidal among scorpion alpha-neurotoxins and, therefore, serves as a model for clarifying the structural basis for their biological activity and selective toxicity. A set of 29 structures was generated without constraint violations exceeding 0.4 A. These structures had root mean square deviations of 0.49 and 1.00 A with respect to the average structure for backbone atoms and all heavy atoms, respectively. Similarly to other scorpion toxins, the structure of Lqh(alpha)IT consists of an alpha-helix, a three-strand antiparallel beta-sheet, three type I tight turns, a five-residue turn, and a hydrophobic patch that includes tyrosine and tryptophan rings in a "herringbone" arrangement. Positive phi angles were found for Ala50 and Asn11, suggesting their proximity to functionally important regions of the molecule. The sample exhibited conformational heterogeneity over a wide range of experimental conditions, and two conformations were observed for the majority of protein residues. The ratio between these conformations was temperature-dependent, and the rate of their interconversions was estimated to be on the order of 1-5 s(-1) at 308 K. The conformation of the polypeptide backbone of Lqh(alpha)IT is very similar to that of the most active antimammalian scorpion alpha-toxin, AaHII, from Androctonus australis Hector (60% amino acid sequence homology). Yet, several important differences were observed at the 5-residue turn comprising residues Lys8-Cys12, the C-terminal segment, and the mutual disposition of these two regions. 2D NMR studies of the R64H mutant, which is 3 times more toxic than the unmodified Lqh(alpha)IT, demonstrated the importance of the spatial orientation of the last residue side chain for toxicity of Lqh(alpha)IT.

Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic Plasmodium falciparum and the underlying molecular basis.

The antimicrobial activity of various naturally occurring microbicidal peptides was reported to result from their interaction with microbial membrane. In this study, we investigated the cytotoxicity of the hemolytic peptide dermaseptin S4 (DS4) and the nonhemolytic peptide dermaseptin S3 (DS3) toward human erythrocytes infected by the malaria parasite Plasmodium falciparum. Both DS4 and DS3 inhibited the parasite's ability to incorporate [3H]hypoxanthine. However, while DS4 was toxic toward both the parasite and the host erythrocyte, DS3 was toxic only toward the intraerythrocytic parasite. To gain insight into the mechanism of this selective cytotoxicity, we labeled the peptides with fluorescent probes and investigated their organization in solution and in membranes. In Plasmodium-infected cells, rhodamine-labeled peptides interacted directly with the intracellular parasite, in contrast to noninfected cells, where the peptides remained bound to the erythrocyte plasma membrane. Binding experiments to phospholipid membranes revealed that DS3 and DS4 had similar binding characteristics. Membrane permeation studies indicated that the peptides were equally potent in permeating phosphatidylserine/phosphatidylcholine vesicles, whereas DS4 was more permeative with phosphatidylcholine vesicles. In aqueous solutions, DS4 was found to be in a higher aggregation state. Nevertheless, both DS3 and DS4 spontaneously dissociated to monomers upon interaction with vesicles, albeit with different kinetics. In light of these results, we propose a mechanism by which dermaseptins permeate cells and affect intraerythrocytic parasites.

The principal neutralizing determinant of HIV-1 located in V3 of gp120 forms a 12-residue loop by internal hydrophobic interactions.

The interactions of the peptide RP135a (RKSI-RIQRGPGRAFVT), corresponding to residues 311-326 of gp120 of HIV-1IIIB, with the anti-gp120 HIV-1IIIB neutralizing antibody 0.5 beta were studied by NMR. The NOESY difference spectra measured using specifically deuterated derivatives of the peptide show exclusively the interactions of the deuterated residues both within the bound peptide and with the Fab fragment of the antibody. These measurements reveal hydrophobic interactions within the bound peptide between Ile-4, Ile-6 and Val-15 that create a 12-residue loop with these residues at the base and the conserved GPGR sequence at its top.

NMR mapping of the antigenic determinant recognized by an anti-gp120, human immunodeficiency virus neutralizing antibody.

The 24-amino-acid peptide RP135 (NNTRKSIRIQRGPGRAFVTIGKIG) corresponds in its amino acid sequence to the principal neutralizing determinant of the human immunodeficiency virus type-1, IIIB isolate (HIV-1IIIB, residues 308-331 of the envelope glycoprotein gp120). In order to map the antigenic determinant recognized by 0.5 beta, the complex of RP135 with an anti-gp120 HIV neutralizing antibody, 0.5 beta, which cross reacts with the peptide, was studied by using two-dimensional NMR spectroscopy. A combination of homonuclear Hartmann Hahn two-dimensional experiment and roating-frame Overhauser enhancement spectroscopy of the Fab/peptide complex measured in H2O was used to eliminate the resonances of the Fab and the tightly bound peptide residues and to obtain sequential assignments for those parts of the peptide which retain considerable mobility upon binding. In this manner, a total of 14 residues (Ser6-Thr19) were shown to be part of the antigenic determinant recognized by the antibody 0.5 beta. Lys5 and Ile20 were found to retain considerable mobility in the bound peptide while their amide protons undergo significant change in chemical shift upon binding. This observation suggests that these two residues are at the boundaries of the determinant recognized by the antibody. Competitive binding experiments using truncated peptides strongly support the NMR observations.

Interfacial conformation of dipalmitoylglycerol and dipalmitoylphosphatidylcholine in phospholipid bilayers.

Diacylglycerols are minor constituents of membrane lipids, yet are essential in the activation and membrane association of protein kinase C. Solid-state 13C NMR experiments have been used to characterize the orientation of the glycerol backbone of dipalmitoylglycerol (DPG) and dipalmitoylphosphatidylcholine (DPPC) in egg phosphatidylcholine (PC) bilayers. The 13C NMR spectra of both DPG and DPPC specifically 13C-labeled at the sn-2 chain carbonyl exhibit a single narrow resonance (approximately 2 ppm) in liquid-crystalline egg PC bilayers. In contrast, specific 13C-labeling of both the sn-1 and sn-2 chain carbonyls results in an additional broad component (24-32 ppm) with an axially symmetric line shape. These data reveal that DPG has a distinct motionally-averaged structure in PC bilayers that is similar to that of DPPC and is not significantly affected by the absence of the large polar PC headgroup. The NMR line shapes are roughly consistent with the results of previous FTIR and NMR studies that indicate the sn-1 chain extends from the C1 carbon of the glycerol backbone into the hydrophobic interior of the bilayer, while the sn-2 chain first extends parallel to the bilayer surface and incorporates a bend at the ester linkage in order to keep the sn-1 and sn-2 chains parallel. However, the data suggest that the time-averaged orientation of the glycerol backbone is tilted from the bilayer normal, in contrast to the nearly parallel orientation observed in the crystal structures of phosphatidylcholines and phosphatidylethanolamines or the perpendicular orientation observed in the crystal structures of diacylglycerols.