Transition temperatures in the sensitivity of escherichia coli B/r to lysozyme

Lysozyme attacked Escherichia coli B/r in the absence of EDTA or imposed osmotic shocks when the cells were rapidly cooled below specific temperatures. Cells subjected to lysozyme while being cooled to below 20°C began to lose ability to subsequently form colonies. This sensitivity increased with decreasing temperatures and almost all cells cooled to 0°C were affected. Slightly hypertonic solutions did not improve survival. Cells cooled first to as low as 5°C and then subjected to lysozyme while cool did not lose their ability to form colonies subsequent to rewarming. However, 70% of the cells cooled first to 0°C and subjected to lysozyme lost their colony-forming ability. Cell lysis also began when treated near 5°C, but even when treated at 0°C about 50% of the cells maintained their rod shape in the presence of lysozyme. These results are discussed in terms of a possible phase transition in a portion of the cell envelope and/or a transient osmotic swelling as a results of metabolic pumps failing at the low temperatures.     

微波杀菌过程中大肠杆菌与金黄色葡萄球菌细胞膜通透性的改变

对细胞膜通透性变化的研究是认识微波杀菌机理的途径之一。用荧光探针检测微波处理后细胞内Ca2 浓度的变化,可以精确地表征细胞膜通透性的改变。选用二乙酸荧光素(FDA)和Fluo-3/AM两种荧光染料,对大肠杆菌(Escherichiacoli)和金黄色葡萄球菌(Staphylococcus aureus)经微波处理后的酯酶活性及细胞膜通透性进行研究,结果表明大肠杆菌与金黄色葡萄球菌的胞内非特异性酯酶(NSE)活性及细胞膜通透性的变化情形有所不同。在50℃、55℃、60℃和65℃微波处理条件下,大肠杆菌细胞膜通透性分别增加了20.7%、28.1%、74.8%、89.8%,而金黄色葡萄球的增加不显著,分别比对照组提高了4.1%、6.0%、21.9%和19.7%。细胞膜通透性的改变与微生物致死率有一定的相关性,也可能是微波杀菌非热效应的表现之一。     

On the ordering of N-alkane and N-alcohol solutes in phospholipid bilayer model membrane systems

²H-NMR measurements of the ordering of several n-alkane and one n-alcohol solutes in lipid bilayers formed from dimyristoyl lecithin (DML) are reported. The results are consistent with orientation of the solutes between the lipid chains, the n-alkanes having a preference for the bilayer interior, while the n-octanol is anchored at the bilayer surface. Solubility of the short chain n-alkanes, n-hexane and n-octane as an ordered component in the L_α phase is more limited than that of n-dodecane. The NMR data are supported by low angle X-ray diffraction results which confirm that n-octanol has a more dramatic influence on bilayer area than the n-alkanes.     

PhoE protein pore of the outer membrane of Escherichia coli K12 is a particularly efficient channel for organic and inorganic phosphate

This study was undertaken to investigate the proposed in vivo pore function of PhoE protein, an Escherichia coli K12 outer membrane protein induced by growth under phosphate limitation, and to compare it with those of the constitutive pore proteins OmpF and OmpC. Appropriate mutant strains were constructed containing only one of the proteins PhoE, OmpF or OmpC, or none of these proteins at all. By measuring rates of nutrient uptake at low solute concentrations, the proposed pore function of PhoE protein was confirmed as the presence of the protein facilitates the diffusion of P_i through the outer membrane, such that a pore protein deficient strain behaves as a K_m mutant. Comparison of the rates of permeation of P_i, glycerol 3-phosphate and glucose 6-phosphate through pores formed by PhoE, OmpF and OmpC proteins shows that PhoE protein is the most effective pore in facilitating the diffusion of P_i and phosphorus-containing compounds. The three types of pores were about equally effective in facilitating the permeation of glucose and arsenate. Possible reasons for the preference for P_i and P_i-containing solutes are discussed.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

The effects of weak acids on potassium uptake by Escherichia coli K-12. Inhibition by low cytoplasmic pH

The activity of the Escherichia coli K⁺ transport system TrkA was measured as a function of the cytoplasmic pH of the cell. For this purpose, pH_in was decreased by the addition of the weak acids acetic acid, benzoic acid or salicylic acid to K⁺-depleted cells. Under these conditions, the initial rate of K⁺ uptake decreased strongly with pH_in, and was almost independent of the acid used. This inhibition was due to a strong decrease in the V_max for K⁺ uptake, which indicates that low cytoplasmic pH inactivates the TrkA K⁺ uptake system. The relevance of this inhibition for growth and metabolism at low pH_in is discussed.     

大肠杆菌细胞裂解系统的构建及其在真菌毒素降解酶表达中的应用

目的:大肠杆菌中分泌表达重组蛋白受限于其分泌效率,为此设计构建大肠杆菌诱导裂解系统以实现胞内重组蛋白的快速高效分泌。方法:利用大肠菌素E7对细胞的裂解能力,构建共表达目标重组蛋白和E7的大肠杆菌细胞裂解系统,使目标重组蛋白在E7表达后得以释放到培养基中。结果:首先以红色荧光蛋白(red fluorescent protein,RFP)为报告基因,在pET28a(+)载体上构建大肠杆菌素E7和红色荧光蛋白两个表达盒,通过对比分析IPTG一步诱导和IPTG-阿拉伯糖分步诱导系统蛋白质的表达效果,发现分步诱导系统能够更高效地表达并释放目标蛋白到培养基。在IPTG-阿拉伯糖分步诱导裂解系统中表达玉米赤霉烯酮降解酶基因,培养基上清液中检测到玉米赤霉烯酮降解酶有较好的表达量和较高的活性,能够在37℃反应30min的条件下降解约5. 8μg玉米赤霉烯酮毒素。结论:利用大肠菌素E7成功构建大肠杆菌细胞裂解系统,并且此系统在快速释放胞内表达外源蛋白方面有适用性。     

The proton pore in the Escherichia coli F0F1-ATPase: A requirement for arginine at position 210 of the a-subunit

Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F₀ portion of the F₀F₁-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F₀F₁-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F₁-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F₀ (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62–69).     

Thermotropism and hydration properties of POPE and POPE-cholesterol systems as revealed by solid state2H and31P-NMR

The partial phase diagram and the hydration properties of the 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)-water system, in the absence and presence of 30 mol% cholesterol, have been investigated by solid state phosphorus NMR of the lipid and deuterium NMR of heavy water. The POPE-D₂O phase diagram resembles other phosphatidylethanolamine (PE)-water systems: below water-to-lipid molar ratios (R_i) of 3 the lamellar gel (L or L_c)-to-hexagonal type II (H_II) phase sequence is observed on increasing the temperature. For R_i3 the thermotropic sequence (L or L_c)-L-H_II is detected. On increasing hydration from R_i=3, the H_II phase is detected from 40°C to 85°C whereas the gel phase is observed from 40°C to 30°C. The limiting hydrations of the gel, L and H_II phases are R_i 3, 17 and 20, respectively. The number of bound water molecules per lipid is ca. 8 in both the La and HII phases. The presence of cholesterol stabilizes the hexagonal phase 20°C below temperatures at which it is observed in its absence and reduces the limiting hydration of the fluid and hexagonal phases to Ri 9 and 14, respectively. The structure and/or dynamics of the water bound to the interface are markedly modified on going from the L to the H_II phase.Abbreviations NMR Nuclear magnetic resonance - DDPE 1,2-Didodecyl-rac-glycerol-3-phosphoethanol-amine - DHPE 1,2-Dihexadecyl-sn-glycerol-3-phosphoethanol-amine - DOPE 1,2-Dioleoyl-sn-glycerol-3-phosphoethanol-amine - POPE 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoetha-nolamine - DAPE 1,2-Diarachinoyl-sn-glycerol-3-phosphoethanol-amine - DMPC 1,2-Dimyristol-sn-glycerol-3-phosphocholine - DPPC 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine - T_c lamellar gel-to-lamellar fluid transition temperature - T_h lamellar fluid-to-hexagonal transition temperature     

Cloning of the nitrile hydratase gene from Nocardia sp. in Escherichia coli and Pichia pastoris and its functional expression using site-directed mutagenesis

To obtain a recombinant Rhodococcus or Nocardia with not only higher enzymatic activity but also better operational stability and product-tolerance ability for bioconversion of acrylamide from acrylonitrile, an active and stable expression system of nitrile hydratase (NHase) was tried to construct as the technical platform of genetic manipulations. Two NHase genes, NHBA and NHBAX, from Nocardia YS-2002 were successfully cloned, based on bioinformatics design of PCR primers, and inserted into plasmid pUC18 and pET32a, respectively. Then, two recombinant Escherichia coli strains, JM105 (pUC18-NHBA) and BL21 (DE3) (pET32a-NHBAX) were constructed and their expressions of NHase were focused. The induction results showed that there was either no NHase activity in JM105 (pUC18-NHBA), or as low as 0.04 U (1 U=1 μmol acrylamide min⁻¹ mg⁻¹ dry cell) in BL21 (DE3) (pET32a-NHBAX). SDS-PAGE results showed that the -subunit of NHBA and NHBAX could not be efficiently expressed in both recombinant E. coli strains. The novel Pichia pastoris system was also applied to express NHase, but the expression level remained quite low (0.5–0.6 U) and the protein was unstable. For solving this problem, a possible genetic strategy, site-directed mutagenesis of the -subunit of the NHase was carried out. After the successful mutagenesis of the original rare start codon gtg into atg, a new recombinant strain, E. coli XL1-Blue (pUC18-NHBA^M), was screened and the NHase activity stably reached as high as 51 U under the same induction conditions.     

Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing

Freeze-thawing of Escherichia coli cells caused a release of cell membrane components such as protein, phospholipids and lipopolysaccharides. A greater amount of release and a lesser extent of cell survival were seen in slow freeze-thawing than in rapid freeze-thawing. Several dehydrogenases in the cells were also freed. The mode of release was also dependent on the rate of freeze-thawing.The materials released by slow freeze-thawing were found to be mostly composed of outer membrane components, whereas the materials released by rapid freeze-thawing contained cytoplasmic as well as outer membrane components. The chemical composition of these fragments differed significantly from that of the original membranes. The relative content of cytoplasmic membrane-bound enzymes in these fragments also differed from that of the cytoplasmic membrane.The fragmentation was assumed to have resulted mainly from the crystallization of external water. In slow freeze-thawing, it was considered that the phase separation of the membrane phospholipid bilayer increased the possibility of outer membrane fragmentation. Rapid freeze-thawing caused cytoplasmic membrane damage to the cells as well as to the outer membrane. In rapid freeze-thawing, the effect of phase separation appeared to be small because of rapid passage through the transition temperatures.The presence of 10% glycerol completely inhibited the release of cellular materials and enzymes. Cell survival was maintained at a high level in the glycerol-treated samples whether freeze-thawed slowly or rapidly.     

Architecture of the outer membrane of Escherichia coli K12 IV. Relationship between outer membrane particles and aqueous pores

The hypothesis that intramembraneous particles, observed in the outer membrane of Escherichia coli by freeze-fracture electron microscopy, are the morphological representation of aqueous pores, was tested. A mutant which is deficient in five major outer membrane proteins, b, c, d, e and the phage λ receptor protein, contains a largely decreased number of intramembraneous particles and also shows a greatly decreased rate of uptake of several solutes. In derivatives of this strain which contain only one of these proteins in large amounts a strong decrease of the number of intramembraneous particles is observed, which is accompanied by a complete restoration of the rate of uptake of those solutes which use pores in which the protein in question is involved. The results provide strong evidence for the notion that an individual pore contains only one protein species, a property which has been found earlier for individual particles. The observed correlation between particles and aqueous pores strongly supports the hypothesis that the particles are the morphological representation of pores. Implications of this hypothesis for the structure of the particles are discussed.     

Fuctional organization of the outer membrane of escherichia coli: Phage and colicin receptors as components of iron uptake systems

The functional interaction of outer memberane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 anf ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all colicins. The interaction of the ton A, Ton B, and feu functions apparently permits quite different “substrates” to overcome the permeablility barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of ³H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferriferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source waw used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: (1) ton A; (2) ton B mutants; (3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; (4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; (5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000–83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene. The nature of these proteins and their possible role in iron transport is disussed.     

CuB promotes both binding and reduction of dioxygen at the heme-copper binuclear center in the Escherichia coli bo-type ubiquinol oxidase

A Cu_B-deficient mutant of the Escherichia coli bo-type ubiquinol oxidase exhibits a very low oxidase activity that is consistent with a decreased dioxygen binding rate. During the turnover, a photolabile reaction intermediate persists for a few hundred milliseconds, due to much slower heme o-to-ligand electron transfer. Thus, the lack of Cu_B seems to have endowed the mutant enzyme with myoglobin-like properties, thereby stabilizing the CO-bound form, too. Accordingly we conclude that Cu_B plays a pivotal role in preferential trapping and efficient reduction of dioxygen at the heme-copper binuclear center.     

The spacing of Escherichia coli DNA gyrase sites cleaved in vivo by treatment with oxolinic acid and sodium dodecyl sulfate

In an attempt to locate gyrase binding sites in a specific region of the chromosome of E. coli, we have reinvestigated gyrase-promoted cleavage of chromosomal DNA by oxolinic acid and sodium dodecyl sulfate. Contrary to a previous report suggesting the presence of one site every 100 kb of DNA (Snyder and Drlica, J. Mol. Biol. 131, 287-302), we found frequencies of one cleavage every 25 or 12 kb depending on the growth medium. A search for cleavage sites by Southern blot hybridization failed to reveal any binding site cleaved at a high frequency. These results suggest that the actual spacing of sites is much closer than that determined from the frequency of cleavage. Measurement of the average size of fragments containing defined DNA sequences indicated that the frequency of sites varies along the chromosome. The region located opposite to oriC carries relatively few sites.     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB⁺ gene of the F plasmid, rifampin, added at 42°C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257–258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784–789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolidigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (M_r 12 000) newly synthesized at 42°C in the presence of rifampin appeared to be the product of the srnB⁺ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30°C, or the addition of Mg²⁺ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB⁺ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB⁺ gene.     

The relationships between growth temperature,fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37°C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37°C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73–76%), phosphatidylglycerol (9–11%) and cardiolipin (11–13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37°C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at ?13, ?9 and 1°C for membranes from cells grown at 5, 22 and 37°C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5°C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.     

Effect of feeding sun-dried seaweed (Ascophyllum nodosum) on fecal shedding of Escherichia coli O157:H7 by feedlot cattle and on growth performance of lambs

Thirty-two steers orally inoculated with a four-strain mixture (10¹⁰ CFU) of nalidixic acid-resistant Escherichia coli O157:H7 had sun-dried Ascophyllum nodosum seaweed (Tasco-14™) added to their barley-based diet (860 g/kg barley grain and 90 g/kg whole crop barley silage, dry matter basis) to assess its effectiveness in reducing fecal shedding of the pathogen. Steers were housed in four groups of eight and received Tasco-14™ in the diet, in place of barley, at levels (as fed) of 10 g/kg for 14 days (T1-14), 20 g/kg for 7 days (T2-7), 20 g/kg for 14 days (T2-14), or not at all (i.e., control, CON). The dietary treatments commenced 7 days after E. coli O157:H7 inoculation and fecal shedding patterns were examined over 14 weeks. Water, water–trough interface, feed and fecal pat samples were also collected weekly and cultured for E. coli O157:H7. Detection of the pathogen in fecal samples was less frequent (P<0.05) in T1-14 (99/168) and T2-7 (84/168) versus CON (135/168) and T2-14 (115/168), and the concentrations of E. coli O157:H7 recovered in feces from T1-14 and T2-7 steers were lower (P<0.005) than from CON or T2-14 steers. Rates of decline in shedding of E. coli O157:H7 were similar among treatments, but final numbers of E. coli O157:H7 were lower (P<0.05) in T1-14 and T2-7 as compared to T2-14 and CON. Fecal volatile fatty acid concentrations and pH were similar among treatments, suggesting no fecal alterations that were antagonistic to survival. E. coli O157:H7 was present in 1 (from T2-7) of 56 cattle drinking water samples, 2 of 56 (T1-14, CON) feed samples and 32 of 56 fecal pats. A second experiment investigated effects of the dietary treatment on growth performance of non-inoculated sheep. Tasco-14™ was administered to 40 individually fed Canadian Arcott lambs beginning at day 56 of a 105-day finishing period. The lambs received Tasco-14™ at 0 g/kg (control, CON), at 10 g/kg for 14 days (T1-14), 20 g/kg for 14 days (T2-14), 10 g/kg for 28 days (T1-28) or at 20 g/kg for 7 days (T2-7) as a top-dress on their pelleted, barley grain-based diet (n = 8). E. coli O157:H7 was not isolated from fecal samples collected at 4-week intervals, but generic E. coli populations were lower (P<0.05) in T1-28 lambs than in other treatments. Average daily gain, feed intake, feed efficiency and carcass traits did not differ among treatments. Our challenge study supports past studies showing that Tasco-14™ decreases shedding of E. coli O157:H7 by cattle. The lamb study shows that this additive did not directly affect feed intake or animal growth.     

3D-Structure of the interior fusion peptide of HGV/GBV-C by 1H NMR, CD and molecular dynamics studies

In this work, we present a structural characterization of the putative fusion peptide E2(279-298) corresponding to the E2 envelope protein of the HGV/GBV-C virus by (1)H NMR, CD and MD studies performed in H(2)O/TFE and in lipid model membranes. The peptide is largely unstructured in water, whereas in H(2)O/TFE and in model membranes it adopts an helical structure (approximately 65-70%). The partitioning free energy DeltaG ranges from -6 to -7.5 kcal mol(-1). OCD measurements on peptide-containing hydrated and oriented lipid multilayers showed that the peptide adopts a predominantly surface orientation. The (1)H NMR data (observed NOEs, deuterium exchange rates, Halpha chemical shift index and vicinal coupling constants) and the molecular dynamics calculations support the conclusions that the peptide adopts a stable helix in the C-terminal 9-18 residues slightly inserted into the lipid bilayer and a major mobility in the amino terminus of the sequence (1-8 residues).