Structural comparison in solution of a native and retro peptide derived from the third helix of Staphylococcus aureus protein A,domain B: retro peptides,a useful tool for the discrimination of helix stabilization factors dependent on the peptide chain orientation

A peptide fragment corresponding to the third helix of Staphylococcus Aureus protein A, domain B, was chosen to study the effect of the main‒chain direction upon secondary structure formation and stability, applying the retro‒enantio concept. For this purpose, two peptides consisting of the native (Ln) and reversed (Lr) sequences were synthesized and their conformational preferences analysed by CD and NMR spectroscopy. A combination of CD and NMR data, such as molar ellipcitity, NOE connectivities, Hα and NH chemical shifts, ³J_αN coupling constants and amide temperature coefficients indicated the presence of nascent helices for both Ln and Lr in water, stabilized upon addition of the fluorinated solvents TFE and HFIP. Helix formation and stabilization appeared to be very similar in both normal and retro peptides, despite the unfavourable charge–macrodipole interactions and bad N-capping in the retro peptide. Thus, these helix stabilization factors are not a secondary structure as determined for this specific peptide. In general, the synthesis and confirmational analysis of peptide pairs with opposite main‒chain directions, normal and retro peptides, could be useful in the determination of secondary structure stabilization factors dependent on the direction. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of topology, length, and charge on the activity of a kininogen-derived peptide on lipid membranes and bacteria

Effects of topology, length, and charge on peptide interactions with lipid bilayers was investigated for variants of the human kininogen-derived peptide HKH20 (HKHGHGHGKHKNKGKKNGKH) by ellipsometry, CD, fluorescence spectroscopy, and z-potential measurements. The peptides display primarily random coil conformation in buffer and at lipid bilayers, and their lipid interaction is dominated by electrostatics, the latter evidenced by higher peptide adsorption and resulting membrane rupture for an anionic than for a zwitterionic membrane, as well as by strongly reduced adsorption and membrane rupture at high ionic strength. At sufficiently high peptide charge density, however, electrostatic interactions contribute to reducing the peptide adsorption and membrane defect formation. Truncating HKH20 into overlapping 10 amino acid peptides resulted in essentially eliminated membrane rupture and in a reduced amount peptide charges pinned at the lipid bilayer. Finally, cyclic HKH20 was found to be less efficient than the linear peptide in causing liposome rupture, partly due to a lower adsorption. Analogous results were found regarding bactericidal effects.     

Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying

Freeze-drying is a process commonly used in starter culture preparation. To improve the survival rate of bacteria during the process, cryoprotectives are usually added before freezing. This study investigated the influence of the addition of sucrose, fructo-oligosaccharides (FOS), inulin and skim milk on the viability and membrane integrity of Lactobacillus reuteri TMW1.106 during freezing, freeze-drying and storage. The effect of drying adjuncts on survival was correlated to their interaction with bacterial membrane by determination of the parameters membrane fluidity and membrane lateral pressure. Sucrose, FOS and skim milk significantly enhanced survival of exponential-phase cells of L. reuteri during freeze-drying. Cellular viability during storage of exponential-phase cells remained highest for cells dried in the presence of skim milk and inulin. Membranes of these cells were completely permeabilized after freeze-drying. The application of FOS significantly improved survival of stationary phase cells of L. reuteri TMW1.106 after freeze-drying and storage. This increased viability of L. reuteri TMW1.106 in the presence of FOS correlated to improved membrane integrity. Fructo-oligosaccharides and fructans, but not gluco-oligosaccharides interacted with membrane vesicles prepared from L. reuteri TMW1.106 as indicated by increased membrane lateral pressure in the presence of FOS and fructans. Increased membrane integrity of stationary phase L. reuteri TMW1.106 was attributed to direct interactions between FOS and the membrane which leads to increased membrane fluidity and thus improved stability of the membrane during and rehydration.     

Backbone dynamics of membrane proteins in lipid bilayers: the effect of two-dimensional array formation as revealed by site-directed solid-state C NMR studies on [3-C]Ala- and [1-C]Val-labeled bacteriorhodopsin

We have recorded site-directed solid-state ¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled bacteriorhodopsin (bR) as a typical membrane protein in lipid bilayers, to examine the effect of formation of two-dimensional (2D) lattice or array of the proteins toward backbone dynamics, to search the optimum condition to be able to record full ¹³C NMR signals from whole area of proteins. Well-resolved ¹³C NMR signals were recorded for monomeric [3-¹³C]Ala-bR in egg phosphatidylcholine (PC) bilayer at ambient temperature, although several ¹³C NMR signals from the loops and transmembrane α-helices were still suppressed. This is because monomeric bR reconstituted into egg PC, dimyristoylphosphatidylcholine (DMPC) or dipalmytoylphosphatidylcholine (DPPC) bilayers undergoes conformational fluctuations with frequency in the order of 10⁴-10⁵ Hz at ambient temperature, which is interfered with frequency of magic angle spinning or proton decoupling. It turned out, however, that the ¹³C NMR signals of purple membrane (PM) were almost fully recovered in gel phase lipids of DMPC or DPPC bilayers at around 0 °C. This finding is interpreted in terms of aggregation of bR in DMPC or DPPC bilayers to 2D hexagonal array in the presence of endogenous lipids at low temperature, resulting in favorable backbone dynamics for ¹³C NMR observation. It is therefore concluded that [3-¹³C]Ala-bR reconstituted in egg PC, DMPC or DPPC bilayers at ambient temperature, or [3-¹³C]Ala- and [1-¹³C]Val-bR at low temperature gave rise to well-resolved ¹³C NMR signals, although they are not always completely the same as those of 2D hexagonal lattice from PM.     

Influence of Ca on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca-activated K channel

The involvement of Ca²⁺-activated K⁺ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3′-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be −57 ± 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K⁺ concentration, [K⁺]_o, and were significantly enhanced by exogenous calcium. The apparent V_max of Na⁺-dependent glycine uptake was doubled in the presence of calcium, whereas the K_0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca²⁺-activated K⁺ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K⁺]_o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC₅₀ of 9.4 μM; and (3) cells treated with the Ca²⁺-activated K⁺ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca²⁺-activated K⁺ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na⁺-dependent amino acids.     

Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells

Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-l-glycero-d-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP⁺, instead of NAD⁺, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.     

Effects of simultaneous changes in light, nutrients, and herbivory levels, on the structure and function of a subtropical turtlegrass meadow

The interactive effects of light, nutrients, and simulated herbivory on the structure and functioning of a subtropical turtlegrass bed were analyzed monthly from May to October 2001 in Perdido Bay, FL. For each of the three factors, two levels were evaluated in a factorial design with four replicates per treatment. The variables included: light, at ambient and 40% reduction; nutrients, at ambient and 2× ambient concentrations; and herbivory, with no herbivory and simulated effects of a density of 15 sea urchins/m². In practice, light levels turned out to be 40% of surface PAR for ambient conditions, and 16% for shaded plots. Biomass removed as herbivory represented, on average, slightly less than 20% of the above-ground biomass. Separate three-way ANOVAs found no significant three-way interactions for any of the response variables, and few two-way interactions. There were no significant nutrient effects on turtlegrass above-ground biomass, although nutrient additions produced significant decreases in epibiont biomass, and net above-ground primary production (NAPP); significant increases in below-ground biomass during the peak of the growing season. Shoot density and average number of leaves per shoot increased significantly, while the C/N ratio of the oldest leaf in the enriched plots decreased significantly. Light reduction significantly negatively affected all response variables, except below-ground biomass, shoot density and leaf length. Herbivory had isolated and inconsistent significant effects on below-ground biomass, shoot density, average number of leaves per shoot, and leaf length and width. Overall, our results indicate that nutrients are not limiting in Perdido Bay, and that nutrient additions had mostly detrimental effects. Light appeared to be the most important variable limiting seagrasses growth and abundance, and as with terrestrial plants, seagrasses seemed to respond more to light and nutrients than to herbivory. However, it is essential that additional tests of the single and interactive effects of the three key factors of light, nutrients and herbivory be done to evaluate the generality of our work, since our study is the first of its kind in seagrass meadows.     

Plasmodium yoelii: the effect of second blood meal and anti-sporozoite antibodies on development and gene expression in the mosquito vector, Anopheles stephensi

The sporogonic development of the malaria parasite takes place in the mosquito and a wide range of factors modulates it. Among those, the contents of the blood meal can influence the parasite development directly or indirectly through the mosquito response to the infection. We have studied the effect of a second blood meal in previously infected mosquitoes and the effect of anti-sporozoite immune serum on parasite development and mosquito response to the infection. The prevalence and intensity of infection and gene expression of both Plasmodium yoelii and Anopheles stephensi was analyzed. We verified that a second blood meal and its immune status interfere with parasite development and with Plasmodium and mosquito gene expression.