Design of infrared optical absorber using silver nanorings array made by a top-down process.

This paper presents the numerical simulation and fabrication of a metasurface composed of silver nanorings with a split-ring gap. These nanostructures can exhibit optically-induced magnetic responses with unique possibilities to control absorption at optical frequencies. The absorption coefficient of the silver nanoring was optimized by performing a parametric study with Finite Difference Time Domain (FDTD) simulations. The absorption and scattering cross sections of the nanostructures are numerically calculated to assess the impact of the inner and outer radii, the thickness and the split-ring gap of one nanoring, as well as the periodicity factor for a group of four nanorings. This showed full control on resonance peaks and absorption enhancement in the near infrared spectral range. The experimental fabrication of this metasurface made of an array of silver nanorings is achieved by e-beam lithography and metallization. Optical characterizations are then carried out and compared to the numerical simulations. In contrast to usual microwave split-ring resonator metasurfaces reported in literature, the present study shows both the realization by a top-down process and modelling performed in the infrared frequency range.

Early skeletal colonization of the coral holobiont by the microboring Ulvophyceae Ostreobium sp.

Ostreobium sp. (Bryopsidales, Ulvophyceae) is a major microboring alga involved in tropical reef dissolution, with a proposed symbiotic lifestyle in living corals. However, its diversity and colonization dynamics in host's early life stages remained unknown. Here, we mapped microborer distribution and abundance in skeletons of the branching coral Pocillopora damicornis from the onset of calcification in primary polyps (7 days) to budding juvenile colonies (1 and 3 months) growing on carbonate and non-carbonate substrates pre-colonized by natural biofilms, and compared them to adult colonies (in aquarium settings). Primary polyps were surprisingly already colonized by microboring filaments and their level of invasion depended on the nature of settlement substrate and the extent of its pre-colonization by microborers. Growth of early coral recruits was unaffected even when microborers were in close vicinity to the polyp tissue. In addition to morphotype observations, chloroplast-encoded rbcL gene sequence analyses revealed nine new Ostreobium clades (OTU99%) in Pocillopora coral. Recruits and adults shared one dominant rbcL clade, undetected in larvae, but also present in aquarium seawater, carbonate and non-carbonate settlement substrates, and in corals from reef settings. Our results show a substratum-dependent colonization by Ostreobium clades, and indicate horizontal transmission of Ostreobium-coral associations.

Combining high-field EPR with site-directed spin labeling reveals unique information on proteins in action.

In the last decade, joint efforts of biologists, chemists and physicists have helped in understanding the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. In this endeavor, electron paramagnetic resonance (EPR) spectroscopy has played an important role. Characteristic examples of such determining factors are hydrogen-bonding patterns and polarity effects of the microenvironment of protein sites involved in the transfer process. These factors may undergo characteristic changes during the reaction and, thereby, control the efficiency of biological processes, e.g. light-induced electron and proton transfer across photosynthetic membranes or ion-channel formation of bacterial toxins. In case the transfer process does not involve stable or transient paramagnetic species or states, site-directed spin labeling with suitable nitroxide radicals still allows EPR techniques to be used for studying structure and conformational dynamics of the proteins in action. By combining site-directed spin labeling with high-field/high-frequency EPR, unique information on the proteins is revealed, which is complementary to that of X-ray crystallography, solid-state NMR, FRET, fast infrared and optical spectroscopic techniques. The main object of this publication is twofold: (i) to review our recent spin-label high-field EPR work on the bacteriorhodopsin light-driven proton pump from Halobacterium salinarium and the Colicin A ion-channel forming bacterial toxin produced in Escherichia coli, (ii) to report on novel high-field EPR experiments for probing site-specific pK(a) values in protein systems by means of pH-sensitive nitroxide spin labels. Taking advantage of the improved spectral and temporal resolution of high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the transient intermediates of the proteins in biological action is obtained. These intermediates can be observed and characterized while staying in their working states on biologically relevant timescales. The paper concludes with an outlook of ongoing high-field EPR experiments on site-specific protein mutants in our laboratories at FU Berlin and Osnabrück.

Gating movements of colicin A and colicin Ia are different.

Both colicin A and colicin Ia belong to a subfamily of the bacterial colicins that act by forming a voltage-dependent channel in the inner membrane of target bacteria. Both colicin A and Ia open at positive and close at negative potential, but only colicin A exhibits distinctly biphasic turnoff kinetics, implying the existence of two open states. Previous work has shown that Colicin Ia gating is associated with the translocation of a region representing 4 of its alpha helices across the membrane. Also, if its C-terminal, channel-forming domain is detached from the other domains, its N-terminal alpha helix can now also cross the membrane, causing the conductance to drop by a factor of about 6. Colicin A gating also involves the translocation of an internal domain, but we find that its translocated domain is somewhat smaller than that of Ia. Furthermore, while its isolated C-terminal domain can also undergo a transition to a smaller conductance, the conductance change is only about 15%, and the transition does not involve the translocation of the N-terminal alpha helix. Trapping the N-terminus on the cis side prevents neither this small conductance transition nor the biphasic turn-off. So, while the gating of both channels involves large, currently inexplicable conformational changes, these motions are qualitatively different in the two proteins, which may be a reflection of the dissimilar kinetics of closing.

Colicin A immunity protein interacts with the hydrophobic helical hairpin of the colicin A channel domain in the Escherichia coli inner membrane.

The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA). This was inserted into the Escherichia coli inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai). We constructed a series of fusion proteins in which various numbers of sp-pfColA alpha-helices were fused to alkaline phosphatase (AP). We showed that a fusion protein made up of the hydrophobic alpha-helices 8 and 9 of sp-pfColA fused to AP was specifically coimmunoprecipitated with EpCai produced in the same cells. This is the first biochemical evidence that Cai recognizes and interacts with the colicin A hydrophobic helical hairpin.

The C-terminal half of the colicin A pore-forming domain is active in vivo and in vitro.

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of Escherichia coli from the periplasmic side and forms a functional channel. The soluble structure of pfColA consists of a ten-helix bundle containing a hydrophobic helical hairpin. Here, we generated a series of mutants in which an increasing number of sp-pfColA alpha-helices was deleted. These peptides were tested for their ability to form ion channels in vivo and in vitro. We found that the shortest sp-pfColA mutant protein that killed Escherichia coli was composed of the five last alpha-helices of sp-pfColA, whereas the shortest peptide that formed a channel in planar lipid bilayer membranes similar to that of intact pfColA was the protein composed of the last six alpha-helices. The peptide composed of the last five alpha-helices of pfColA generated a voltage-independent conductance in planar lipid bilayer with properties very different from that of intact pfColA. Thus, helices 1 to 4 are unnecessary for channel formation, while helix 5, or some part of it, is important but not absolutely necessary. Voltage-dependence of colicin is evidently controlled by the first four alpha-helices of pfColA.

[How has the management of mentally disabled people changed?]. / Comment l'accueil des handicapés mentaux a-t-il évolué?

Greeting the mentally disabled has largely changed with time. The first attempts to assume "idiots", as Esquirol characterized them, are due to him and his followers, among whom must be mentioned Seguin and Bourneville, whose ward in Bicêtre Hospital was unfortunately suppressed in 1920. A gap appeared then, which private actions tried to fill by founding institutions for mentally disabled children. Nothing has been planned to greet them when grown adults, probably because their life expectancy was then precarious. It is not so at the present time, and their ageing creates a great problem.

Integration of the colicin A pore-forming domain into the cytoplasmic membrane of Escherichia coli.

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherichia coli and apparently formed a functional channel, when generated in vivo. We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments. Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm. We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9. These two domains function independently. Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts. In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening.

Intracellular immunization of prokaryotic cells against a bacteriotoxin.

Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E. coli. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.

Membrane topology of the colicin A pore-forming domain analyzed by disulfide bond engineering.

Four colicin A double-cysteine mutants possessing a disulfide bond in their pore-forming domain were constructed to study the translocation and the pore formation of colicin A. The disulfide bonds connected alpha-helices 1 and 2, 2 and 10, 3 and 9, or 3 and 10 of the pore-forming domain. The disulfide bonds did not prevent the colicin A translocation through the Escherichia coli envelope. However, the mutated colicins were able to exert their in vivo channel activity only after reduction of their disulfide bonds. In vitro studies with brominated phospholipid vesicles and planar lipid bilayers revealed that the disulfide bond that connects the alpha-helices 2 and 10 prevented the colicin A membrane insertion, whereas the other double-cysteine mutants inserted into lipid vesicles. The disulfide bonds that connect either the alpha-helices 1 and 2 or 3 and 10 were unable to prevent the formation of a conducting channel in presence of membrane potential. These results indicate that alpha-helices 1, 2, 3, and 10 remain at the membrane surface after application of a membrane potential.

The channel domain of colicin A is inhibited by its immunity protein through direct interaction in the Escherichia coli inner membrane.

A bacterial signal sequence was fused to the colicin A pore-forming domain: the exported pore-forming domain was highly cytotoxic. We thus introduced a cysteine-residue pair in the fusion protein which has been shown to form a disulfide bond in the natural colicin A pore-forming domain between alpha-helices 5 and 6. Formation of the disulfide bond prevented the cytotoxic activity of the fusion protein, presumably by preventing the membrane insertion of helices 5 and 6. However, the cytotoxicity of the disulfide-linked pore-forming domain was reactivated by adding dithiothreitol into the culture medium. We were then able to co-produce the immunity protein with the disulfide linked pore-forming domain, by using a co-immunoprecipitation procedure, in order to show that they interact. We showed both proteins to be co-localized in the Escherichia coli inner membrane and subsequently co-immunoprecipitated them. The interaction required a functional immunity protein. The immunity protein also interacted with a mutant form of the pore-forming domain carrying a mutation located in the voltage-gated region: this mutant was devoid of pore-forming activity but still inserted into the membrane. Our results indicate that the immunity protein interacts with the membrane-anchored channel domain; the interaction requires a functional membrane-inserted immunity protein but does not require the channel to be in the open state.

Quantification of group A colicin import sites.

Pore-forming colicins are soluble bacteriocins which form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, these colicins first bind to a receptor located on the outer membrane and then are translocated through the envelope. Colicins are subdivided into two groups according to the envelope proteins involved in their translocation: group A colicins use the Tol proteins; group B colicins use the proteins TonB, ExbB, and ExbD. We have previously shown that a double-cysteine colicin A mutant which possesses a disulfide bond in its pore-forming domain is translocated through the envelope but is unable to form a channel in the inner membrane (D. Duché, D. Baty, M. Chartier, and L. Letellier, J. Biol. Chem. 269:24820-24825, 1994). Measurements of colicin-induced K+ efflux reveal that preincubation of the cells with the double-cysteine mutant prevents binding of colicins of group A but not of group B. Moreover, we show that the mutant is still in contact with its receptor and import machinery when it interacts with the inner membrane. From these competition experiments, we conclude that each Escherichia coli cell contains approximately 400 and 1,000 colicin A receptors and translocation sites, respectively.

A single amino acid substitution can restore the solubility of aggregated colicin A mutants in Escherichia coli.

Mutants of colicin A have been prepared in which the three tryptophan residues (Trp86, Trp130 and Trp140), localized in the C-terminal domain of the soluble wild-type protein, have been substituted by phenylalanine. The Trp140Phe single mutation had the effect of decreasing the percentage of protein that is expressed as insoluble aggregates. The created hydrophobic cavity decreased the stability of the protein during its folding, resulting in partial aggregation in the cytoplasm of the Escherichia coli-producing cells. Aggregation was increased when Trp140 was substituted by Lys, Leu or Cys, or if the Trp140 mutation was combined with the Trp86Phe and/or Trp130Phe mutations. A single mutation, Lys113Phe, however, was able to restore the solubility of the aggregated mutants in vivo. Detailed analysis of the 3-D structure of the C-terminal domain of colicin A suggests that filling of the hydrophobic cavity is responsible for this effect.

Unfolding of colicin A during its translocation through the Escherichia coli envelope as demonstrated by disulfide bond engineering.

Three double cysteine mutants, each possessing a disulfide bond in its pore-forming domain, were used to study the translocation of colicin A through the Escherichia coli envelope. These mutated colicins were able to exert their in vivo channel activity only after their disulfide bonds had been reduced by dithiothreitol. In solution, the reduction of the disulfide bonds by dithiothreitol was a slow process whose kinetics depended on the position of the disulfide bond (t1/2 varying between 35 and 100 s). This t1/2 was strongly decreased (t1/2 = 8-9 s) upon predenaturation of the mutated colicins with urea. The t1/2 values of reduction of the mutants bound to E. coli-sensitive cells were similar to those of predenatured colicins. This suggested that the interaction of the oxidized double cysteine mutants with the E. coli envelope triggered their unfolding. The disulfide bonds did not prevent but delayed the translocation of the colicins. The amplitude of the delay and the time at which it occurred during translocation depended on the position of the disulfide bond. We could discriminate between the delays accumulated during binding to the receptor and those during the translocation via OmpF and the Tol proteins.

Uncoupled steps of the colicin A pore formation demonstrated by disulfide bond engineering.

Four disulfide bonds were engineered into the pore-forming domain of colicin A to probe the conformational changes associated with its membrane insertion and channel formation. The soluble pore-forming domain consists of 10 alpha-helices with two outer layers (helices 1, 2, and 3-7, respectively) sandwiching a middle layer of three helices (8-10). Helices 8 and 9 form a hairpin which is completely buried and consists of hydrophobic and neutral residues only. This helical hairpin has been hypothesized to be the membrane anchor. Each double-cysteine mutant possessing an individual disulfide bond, cross-linking either helices 1 to 9 (H1/H9), 5 to 6 (H5/H6), 7 to 8 (H7/H8), or 9 to 10 (H9/H10), respectively, is unable to promote K+ efflux from sensitive Escherichia coli cells. Activity can be restored by addition of a reducing agent. In vitro studies with brominated lipid vesicles and planar lipid bilayers show that the disulfide bond which connects the helices 1 to 9 prevents colicin A membrane insertion, whereas the other disulfide bond mutants insert readily into lipid vesicles. All of the engineered bridges prevented the formation of a conducting channel in the presence of a membrane potential. This novel approach indicates that membrane insertion and channel formation are two separate steps. Moreover, the effects of the distance constraints introduced by the different disulfide bonds on colicin A activity indicate that the helical pair 1 and 2 moves away from the other helices upon membrane insertion. Helices 3-10 remain associated together. As a consequence, the results imply that the helical hairpin lies parallel to the membrane surface. In contrast, induction of the colicin channel by the membrane potential requires a profound reorganization of the helices association. These results are discussed in light of several proposed models of the membrane-bound colicin and channel structures.

The role of electrostatic charge in the membrane insertion of colicin A. Calculation and mutation.

The bacterial toxin colicin A binds spontaneously to the surfaces of negatively charged membranes. The surface-bound toxin must subsequently, however, become an acidic 'molten globule' before it can fully insert into the lipid bilayer. Clearly, electrostatic interactions must play a significant role in both events. The electrostatic field around the toxin in solution was calculated using the finite-difference Poisson-Boltzmann method of the Delphi programme and the known X-ray structure. A large positively charged surface was identified which could be involved in the binding of colicin to negatively charged membranes. The applicability of the result was tested by also calculating the fields around modelled structures of the closely related colicins B and N. Surprisingly, colicin N showed a similar charge distribution in spite of its isoelectric point of pI 10.20 (colicin A has pI 5.44). One reason for this is the strong conservation of certain negative charges in all colicins. There is a single highly conserved aspartate residue (Asp78) on the positively charged face which provides a small but discrete region of negative charge. This residue, Asp78, was replaced by asparagine in the mutant D78N. D78N binds faster to negatively charged vesicles but inserts only half as fast as the wild-type protein into the membrane core. This indicates that, first, the initial membrane binding has a significant electrostatic component and, second, that the isolated charge on Asp78 plays a role in the formation of the insertion intermediate.

Fluorescence energy transfer distance measurements. The hydrophobic helical hairpin of colicin A in the membrane bound state.

The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this study fluorescence resonance energy transfer spectroscopy was used to determine the position of this helical hairpin in the membrane bound state. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulpho-1-naphthyl)ethylenediamine (I-AEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Five mutants I26C (helix 1), F105C (between helices 4 and 5), G166CJ (helix 8), A169C (helix 8-9), G176C (helix 9) were used. All mutants show wild-type binding activity to phosphatidylglycerol vesicles as judged by fluorescence polarization anisotropy, emission wavelength changes and brominated lipid quenching. The three tryptophan residues were used as a compound donor to AEDANS in resonance energy transfer distance determinations. The distances obtained for the soluble form were equal to those found in the crystal structure. On adding vesicles under conditions where intermolecular transfer was avoided the indicated distances increased; I26(10.9 A) F105(3.4 A), G166(3.3 A), A169(1.9 A) and G176(2.9 A). This confirms that, in the absence of a membrane potential, helices 1 and 2 open out onto the membrane surface whilst the helical hairpin remains closely packed against the rest of the structure. The insertion of this hairpin is thus not the driving force behind colicin membrane binding.

[Consent to adoption]. / Le consentement à l'adoption.

Many legal provisions exist that aim at preventing children from being abandoned. However, in some instances, these apparently beneficial measures result in de facto abandonments. In such cases, the abandonment is delayed, and as a result the child is no longer eligible for adoption. This situation leads to a number of successive changes that are emotionally detrimental to the child. We need prophylactic measures aimed at obtaining the "consent to adoption" from mothers who obviously feel and are incapable of being mothers in the full sense of the term.