Refining membrane penetration by a combination of steady-state and time-resolved depth-dependent fluorescence quenching

Accurate determination of the depth of membrane penetration of a fluorescent probe, attached to a lipid, protein, or other macromolecule of interest, using depth-dependent quenching methodology is complicated by thermal motion in the lipid bilayer. Here, we suggest that a combination of steady-state and time-resolved measurements can be used to generate a static quenching profile that reduces the contribution from transverse diffusion occurring during the excited-state lifetime. This procedure results in narrower quenching profiles, compared with those obtained by traditional measurements, and thus improves precision in determination of the underlying depth distribution of the probe.     

Effect of presowing treatment of spring wheat seeds with wheat germ agglutinin on the chlorophyll content, lectin activity in leaves and nitrogen-fixing capacity of rhizospheric microorganisms

The response of spring wheat and rhizospheric nitrogen-fixing micro-organisms to the presowing treatment of seeds by wheat germ agglutinin was investigated in conditions of green house experiments. It was shown, that exogenous lectin induced the metabolic changes in plants and caused an increase in chlorophyll content and activity of endogenous lectins in the leaves, as well as enhanced accumulation of plants biomass and nitrogen-fixing capacity of the rhizospheric micro-organisms. These results evidence for the considerable role of exogenous lectin as a regulator of growth and development of plants and activity of the nitrogen-fixing microorganisms.     

Distribution and favorable binding sites of pyrroloquinoline and its analogues in a lipid bilayer studied by molecular dynamics simulations

The distribution of 1H-pyrrolo[3,2-h]quinoline (PQ), 11H-dipyrido[2,3-a]carbazole (PC) and 7-azaindole (7AI) at a water/membrane interface has been investigated by molecular dynamics (MD) simulations. The MD study focused on favorable binding sites of the azaaromatic probes across a dipalmitoylphosphatidylcholine (DPPC) bilayer. Our simulations show that PQ and PC are preferably accommodated at the hydrocarbon core of the bilayer below the glycerol moiety. In addition, it is found that the hydrophobic aromatic parts of the probes are located inside a more ordered region of DPPC, consisting of hydrophobic lipid chains. In contrast to PQ and PC, 7AI is characterized by a broad distribution between a DPPC interface and water, so that the three preferable binding sites are found across a water/membrane interface. It is found that in the sequence 7AI-PQ-PC, due to the increase of the number of aromatic rings and, hence, the hydrophobic character of the probes, the depth of the probe localization is gradually shifted deeper inside the hydrocarbon core of the bilayer. We found that the probe-lipid hydrogen-bonding contributes weakly to the favorable localizations of the azaaromatic probes inside the DPPC bilayer, so that the probe localization is mainly driven by electrostatic dipole-dipole and van der Waals interactions.     

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.     

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells

AimsThe goal of this study was to evaluate the influence of γ-irradiation on Ca²⁺-activated K⁺ channel (BK_Ca) function and expression in rat thoracic aorta.Main methodsAortic cells or tissues were studied by the measurement of force versus [Ca²⁺]_i, patch-clamp technique, and RT-PCR.Key findingsStimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K⁺ currents. Paxilline, an inhibitor of BK_Ca channels, decreased outward K⁺ current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K⁺ current density. Paxilline decreased K⁺ current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK_Ca channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca²⁺ transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK_Ca α- and β₁-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished.SignificanceThese results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca²⁺ sensitivity in the early post-irradiation period and a decrease in BK_Ca channel expression in the late post-irradiation period.     

Change of the wheat lectin activity and degree of its interaction with different components of compositions of lectin nature

The wheat lectin hemagglutination activity and degree of its interaction with the bacterium Azotobacter chroococcum T79 and aminosaccharide N-acetyl-D-glucosamin hapten of wheat lectin was studied in laboratory experiments with the purpose of creation of biologic activity compositions of lectin nature for plant growing. It was shown that plant-bacterial compositions encloses the "bacteria+lectin" complex, free lectin and bacterial cells. The addition of aminosaccharide N-acetyl-D-glucosamin to wheat lectin, to the bacterial culture and plant-bacterial composition decreases its hemagglutination activity. The possibility of creation of new complexes in this compositions effected by hapten "lectin+hapten", "lectin+hapten+bacteria", "bacteria+hapten" is under discussion.     

Folding of diphtheria toxin T-domain in the presence of amphipols and fluorinated surfactants: Toward thermodynamic measurements of membrane protein folding

Solubilizing membrane proteins for functional, structural and thermodynamic studies is usually achieved with the help of detergents, which, however, tend to destabilize them. Several classes of non-detergent surfactants have been designed as milder substitutes for detergents, most prominently amphipathic polymers called 'amphipols' and fluorinated surfactants. Here we test the potential usefulness of these compounds for thermodynamic studies by examining their effect on conformational transitions of the diphtheria toxin T-domain. The advantage of the T-domain as a model system is that it exists as a soluble globular protein at neutral pH yet is converted into a membrane-competent form by acidification and inserts into the lipid bilayer as part of its physiological action. We have examined the effects of various surfactants on two conformational transitions of the T-domain, thermal unfolding and pH-induced transition to a membrane-competent form. All tested detergent and non-detergent surfactants lowered the cooperativity of the thermal unfolding of the T-domain. The dependence of enthalpy of unfolding on surfactant concentration was found to be least for fluorinated surfactants, thus making them useful candidates for thermodynamic studies. Circular dichroism measurements demonstrate that non-ionic homopolymeric amphipols (NAhPols), unlike any other surfactants, can actively cause a conformational change of the T-domain. NAhPol-induced structural rearrangements are different from those observed during thermal denaturation and are suggested to be related to the formation of the membrane-competent form of the T-domain. Measurements of leakage of vesicle content indicate that interaction with NAhPols not only does not prevent the T-domain from inserting into the bilayer, but it can make bilayer permeabilization even more efficient, whereas the pH-dependence of membrane permeabilization becomes more cooperative. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain

The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N-terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes that take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N-terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time. Here we report that replacement of the three C-terminal histidine residues, H322, H323, and H372, in triple-R or triple-Q mutants prevents effective translocation of the N-terminus. Introduction of these mutations in the full-length toxin results in decrease of its potency. In the context of isolated T-domain, these mutations cause loss of characteristic conductance in planar bilayers. Surprisingly, these mutations do not affect general folding in solution, protein interaction with the membranes, insertion of the consensus transmembrane helical hairpin TH8-9, or the ability of the T-domain to destabilize vesicles to cause leakage of fluorescent markers. Thus, the C-terminal histidine residues are critical for the transition from the inserted intermediate state to the open-channel state in the insertion/translocation pathway of the T-domain.