Thirty-third Annual Meeting February 12 - 16, 1989 Albert B. Sabin Convention Center Cincinnati, Ohio

1989 Biophysical Journal  
PGLa is a polypeptide antibiotic isolated froTm Xenopu4 taev'" granular glands, which has been proposed to form channels in lipid membranes. In this study, circular dichroism spectroscopy was used to examine the conformation of this molecule in a variety of environments, as a basis for understanding its structural features. In addition, phospholipid binding studies were used to demonstrate features which permit the association of this polypeptide with membranes. In the organic solvents methanol
more » ... and trifluoroethanol, PGLa adopts regular structures that are composed of both helical and sheet-type secondary structures. PGLa also has a substantial solubility in water; but in this environment, it forms an entirely disordered structure. Ultracentrifugation studies have shown that only small amounts of PGLa are bound to neutral phospholipids, but that the portion of the polypeptide that IS bound undergoes a significant conformational change from that in the aqueous solution. Furthermore, if a small amount of negatively charged phospholipid is present in the vesicles, PGLa binding is significantly enhanced, and this binding induces the polypeptide to undergo a transformation from a completely disordered to an almost entirely helical structure. These studies indicate a possible route for the binding and insertion of this polypeptide into membranes, and suggest that a helical channel may be an appropriate model for the basis of its antimicrobial activity. Several outer membrane mutants of Salmonella typhimuriwn have been characterized which possess lipopolysaccharides (LPS) with deficient polysaccharide structures. The Ra mutant is missing the 0-side chain sugars, while the Rb, Rc, Rd" R& and Re mutants have lost a progressively increasing portion of the core region sugars. These mutants are much more susceptible to antibiotics and antimicrobial peptides than the wild-type organism. The effects of altered polysaccharide structure on the structural characteristics of the LPS in the outer membranes of these organisms have received limited attention and remain poorly understood. We have used FT-IR spectroscopy and differential scanning calormetery to monitor the order-todisorder transition of both purified LPS and the intact outer membrane-peptidoglycan complex isolated from a wild-type (smooth) strain (SL 3770) of S. typhimurium and six rough LPS mutant stains (SL 3749, SL 3750, SL 3748, SL 3769, SL 3789, and SL 1102) with the respective LPS chemotypes RZa Rb, Rc, RP, Rdand Re. The fluidity of the outer membranepeptidoglycan complex is similar for all strains examined, with a broad phase change centered slighdy above growth temperature (37°C); however, the thermally-induced phase behavior varies significantly for the extracted LPS as the length of the polysaccharide chain decreases. While the smooth LPS undergoes a very cooperative phase change at 370C, both the transition temperature and degree of cooperativity of phase changes for the rough LPS are decreased, with the exception of the Restrain. The possibility that changes in the LPS molecule may induce altered structural assemblies, including interdigitated or non-lamellar phases, is discussed. Finally, the effects of polymyxin B and the antimicrobial peptide, magainin 2, on the fluidity and structure of the outer membranes and extracted LPS from these organisms is presented. 3-Hydroxybutyrate dehydrogenase (BDH) from bovine heart mitochondria is a membrane-inlaid tetrameric enzyme which requires lecithin for optimal function. Each subunit of BDH has two sulfhydryls, SH-1 and SH-2, which have been selectively derivatized with 2,2,6,6-tetramethylmaleimidopiperidinyl-l-oxyl (MSL). We have compared the accessibility of the MSL nitroxide group at SH-1 and SH-2 to water-soluble reducing agent dithiothreitol (DTT) and to hydrated paramagnetic ions. MSL(SH-1)BDH-lipid complex (50 pM) required exposure to 1 mM DTT to effect reduction of the nitroxide (t½ -2 hr), whereas MSL(SH-2)BDH-MPL was reduced by 0.1 mM DTT more rapidly (t½ -20 min). These results are consistent with studies of nitroxide interaction with paramagnetic ions. The nitroxide at SH-1 interacts by a dipolar mechanism with Mn+ or Gd3+ bound to the lipid vesicle surface. From the dipolar interaction coefficient and MSL size, the distance of SH-1 from the membrane surface was calculated as ; 9 A [Dalton et al., Biochemistry (1987) 26, 2117-2130. The nitroxide of MSL at SH-2 interacts directly with Mn2+ or Gd3+ by a Heisenberg spin exchange mechanism, indicating that SH-2 is close to (s 5 A) the surface. SH-1 is in the vicinity of the active center of BDH, since: (1) its alkylation with MSL decreases the affinity for coenzyme and substrate and diminishes enzyme activity (Vmax reduced -40-fold); and (2) the nitroxide of MSL(SH-1)BDH-lipid is reduced by NADH bound to the enzyme. Alkylation of SH-2 with MSL does not affect the Km for substrate or NAD+, although Vmax is decreased. Hence SH-2 is more distant from the active center. It has been proposed that Acholeplasma laidlawii A accurately regulates the bilayer/nonbilayer phase preference of its membrane lipids by appropriate changes in their polar headgroup distribution. In particular, the proportion of monoglucosyl diacylglycerol (MGDG, the only nonbilayer forning lipid component) appears to decrease with increases in growth temperature, fatty acyl chain unsaturation or cholesterol content, so as to maintain the bilayer/nonbilayer phase transition temperature constant. We have recently investigated the effect of variations in growth temperature, fatty acid composition and cholesterol content on the closely related A. laidlawii B using a much greater range of fatty acid structures and chain lengths and, in each case, we have determined the bilayer/nonbilayer phase transition temperature of the MGDG formed. In contrast to strain A, we find that in A. laidlawii B increases in growth temperature or cholesterol content only reduce MGDG levels in cells enriched in saturated fatty acids, which in any case do not form nonbilayer lipid phases at physiological temperatures. Moreover, we find that there is only a rough relationship between the nonbilayer-fonning tendency of a biosynthetically incorporated exogenous fatty acid and its effect on the ratio of nonbilayer to bilayer preferng lipids in the membane of this organism. In particular, this organism appears to respond in the predicted fashion to branched chain but not to saturated, unsaturated or o-cyclohexyl fatty acids. We therefore conciude that A. laidlawii B does not posses a biosynthetic mechanism which is capable of accurately regulating the bilayer/nonbilayer phase preference of its membrane lipids. . Sphingomyelin (SPM) interacts with cholesterol (chol) more tightly than does PC with similar hydrocarbon chain structure, but the molecular basis for the greater affinity is not fully clear. Increased van der Waals interactions as well as other forces such as hydrogen bonding may contribute to the higher SPM-chol affinity. The hydroxyl group at the 3 position of SPM has been suggested as an additional site at which interaction may occur between SPM and chol. To investigate the influence of the 3-OH group we measured the kinetics of (C-14)chol exchange between SUVs prepared with synthetic SPM analogs bearing substituents at position 3. Donor SUVs contained 10 mol % chol, 75 mol % SPM, and 15 mol % dicetyl phosphate; acceptor SUVs (10-fold excess) contained 10 mol X chol, 90 mol % egg SPM, and (9,10-H-3)-triolein as nonexchangeable marker. Replacement of the 3-OH group by a 3-OMe group gave little change in the half-time for (C-14)chol exchange at 50 C, suggesting that (a) the hydrogen of the 3-OH group does not play an important role as an acceptor in hydrogen bonding with chol, and (b) a moderate increase in steric bulk at this site does not impede SPM-chol interaction. Substitution of the 3-OH group by a 3-0-tetrahydropyranyl group gave a 35-fold enhancement in cholesterol exhange rate, indicating that bulky groups at this position interfere with the molecular packing of chol and SPM in the donor SUVs. The synthesis of 3-substituted SPM analogs and the results of chol exchange rates between SUVs will be presented. Ethanol induced changes in the molecular order of the surface and interior domains of synaptic lasma membranes from genetically selected ethanol sensitive and insensitive mice have been measure using delayed Fourier transform IH-NMR. Ethanol has a disruptive effect in both domains of each strain. The effects are greater in the surface domain for the sensitive strain. From the increase in spectral intensity and a decrease in the apparent linewidth of the choline methyl group, it is postulated that the ethanol causes the average distance between the polar head groups to increase. This is not observed for the insensitive strain. The results will be discussed in relation to the higher monosialoganglioside content in the membranes from the sensitive strain. It is proposed that the basis for the differences in ethanol sensitivity between the two strains arises from the differential ordering at the surface of the neuronal membranes.
doi:10.1016/s0006-3495(89)82822-x fatcat:upklfgq2kzdl7mdg2bi7pqizni