Structural and Functional Characterization of Orcokinin B-like Neuropeptides in the Cuttlefish (Sepia officinalis).

The cuttlefish (Sepia officinalis) is a Cephalopod mollusk that lives in the English Channel and breeds in coastal spawning grounds in spring. A previous work showed that the control of egg-laying is monitored by different types of regulators, among which neuropeptides play a major role. They are involved in the integration of environmental cues, and participate in the transport of oocytes in the genital tract and in the secretion of capsular products. This study addresses a family of neuropeptides recently identified and suspected to be involved in the control of the reproduction processes. Detected by mass spectrometry and immunocytochemistry in the nerve endings of the accessory sex glands of the females and ovary, these neuropeptides are also identified in the hemolymph of egg-laying females demonstrating that they also have a hormone-like role. Released in the hemolymph by the sub-esophageal mass, a region that innervates the genital tract and the neurohemal area of the vena cava, in in vitro conditions these neuropeptides modulated oocyte transport and capsular secretion. Finally, in silico analyses indicated that these neuropeptides, initially called FLGamide, had extensive structural homology with orcokinin B, which motivated their name change.

Contrast-Matched Isotropic Bicelles: A Versatile Tool to Specifically Probe the Solution Structure of Peripheral Membrane Proteins Using SANS.

Obtaining structural information on integral or peripheral membrane proteins is currently arduous due to the difficulty of their solubilization, purification, and crystallization (for X-ray crystallography (XRC) application). To overcome this challenge, bicelles are known to be a versatile tool for high-resolution structure determination, especially when using solution and/or solid state nuclear magnetic resonance (NMR) and, to a lesser extent, XRC. For proteins not compatible with these high-resolution methods, small-angle X-ray and neutron scattering (SAXS and SANS, respectively) are powerful alternatives to obtain structural information directly in solution. In particular, the SANS-based approach is a unique technique to obtain low-resolution structures of proteins in interactions with partners by contrast-matching the signal coming from the latter. In the present study, isotropic bicelles are used as a membrane mimic model for SANS-based structural studies of bound peripheral membrane proteins. We emphasize that the SANS signal coming from the deuterated isotropic bicelles can be contrast-matched in 100% D2O-based buffer, allowing us to separately and specifically focus on the signal coming from the protein in interaction with membrane lipids. We applied this method to the DYS-R11-15 protein, a fragment of the central domain of human dystrophin known to interact with lipids, and we were able to recover the signal from the protein alone. This approach gives rise to new perspectives to determine the solution structure of peripheral membrane proteins interacting with lipid membranes and might be extended to integral membrane proteins.

Two distinct conformational states of Mycobacterium tuberculosis virulent factor early secreted antigenic target 6 kDa are behind the discrepancy around its biological functions.

Early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) are complex proteins secreted by Mycobacterium tuberculosis that play a major role in the pathogenesis of tuberculosis. However, studies focusing on the biological functions of ESAT-6 led to discordant results and the role of ESAT-6 remains controversial. In the present study, we aim to address a potential explanation for this discrepancy and to highlight the physiological impact of two conformational states of ESAT-6. Analysis of a recombinant form of ESAT-6 by native gel electrophoresis, size exclusion chromatography and CD spectroscopy revealed that ESAT-6 forms dimers/multimers with higher molecular weight, which disappeared under the action of the detergent amidosulfobetaine-14 (ASB), giving rise to another conformational state of the protein. NMR has further indicated that ASB-treated versus nontreated ESAT-6 adopted distinct structural forms but with no well defined tertiary structure. However, protein-protein docking analysis favored a dimeric state of ESAT-6. Interestingly, the two preparations presented opposing effects on mycobacterial infectivity, as well as macrophage survival, interferon-γ secretion and membrane pore formation. Thereafter, we generated a recombinant form of the physiological heterodimer ESAT-6/CFP-10 that ASB was also able to dissociate and which showed functions similar to those of ESAT-6 dimers/multimers. Our data suggest that, in the absence of CFP-10, the hydrophobic regions of the ESAT-6 can form dimers/multimers, mimicking the ESAT-6/CFP-10 heterodimer, whereas their dissociation generates a protein presenting entirely different activities. Overall, the present study clarifies the intriguing divergences between reports that could be attributed to the ESAT-6 oligomeric state and sheds light on its importance for a better comprehension of the physiopathology of tuberculosis.

Sterols associated with small unilamellar vesicles (SUVs): intrinsic mobility role for 1H NMR detection.

Small unilamellar vesicles (SUVs) of phospholipids are often used as a membrane model system for studying the interaction of molecules. When using NMR under the standard liquid-state conditions, SUV phospholipid proton spectra can be recorded, exhibiting sharp signals. This is not only because of the fast vesicular tumbling but also because of the combination of this tumbling with the individual motion of the lipids inside the bilayer. This appears evident because addition of cholesterol is responsible of broader resonances because of the slowing down of the lipid motion. On the other hand, no (1)H signal is detected for cholesterol in the bilayer. This lack of detection of the inserted molecules explains why generally SUVs are not considered as a good model for NMR studies under the standard liquid-state conditions. Here, we use two other sterols in order to demonstrate that an increase of the molecular mobility inside the bilayer could allow the detection of their proton resonances. For desmosterol and lanosterol, which show higher mobility inside the bilayer, with increasing lateral diffusion rates, (1)H sterol signals are detected in contrast to cholesterol. For the fast diffusing lanosterol, no significant improvement in detection is observed using deuterated lipids, demonstrating that homonuclear dipolar coupling is fully averaged out. Furthermore, in the case of low mobility such as for cholesterol, the use of a fast magic angle spinning probe is shown to be efficient to recover the full proton spectrum.

Elucidation by NMR solution of neurotensin in small unilamellar vesicle environment: molecular surveys for neurotensin receptor recognition.

Neurotensin (NT) is a tridecapeptide, hormone in the periphery and neurotransmitter in the brain. We used high-resolution nuclear magnetic resonance (NMR) to resolve the three-dimensional structure of NT in a small unilamellar vesicle (SUV) environment. We demonstrate that if the dynamic of the association-dissociation processes of peptide to SUV binding is rapid enough, structural determination can be obtained by solution NMR experiments. Thus, according to the global dynamic of the system, SUVs seem to be an effective model to mimic biological membranes, especially since the lipid composition can be modified or sterols may be added to closely mimic the biological membranes studied. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:2.

Structure and mechanism of action of a de novo antimicrobial detergent-like peptide.

The K4 peptide (KKKKPLFGLFFGLF) was recently demonstrated to display good antimicrobial activities against various bacterial strains and thus represents a candidate for the treatment of multiple-drug resistant infections. In this study, we use various techniques to study K4 behaviour in different media: water, solutions of detergent micelles, phospholipid monolayers and suspension of phospholipid vesicles. First, self-assembly of the peptide in water is observed, leading to the formation of spherical objects around 10nm in diameter. The addition of micelles induces partial peptide folding to an extent depending on the charge of the detergent headgroups. The NMR structure of the peptide in the presence of SDS displays a helical character of the hydrophobic moiety, whereas only partial folding is observed in DPC micelles. This peptide is able to destabilize the organization of monolayer membranes or bilayer liposomes composed of anionic lipids. When added on small unilamellar vesicles it generates larger objects attributed to mixed lipid-peptide vesicles and aggregated vesicles. The absence of calcein leakage from liposomes, when adding K4, underlines the original mechanism of this linear amphipathic peptide. Our results emphasize the importance of the electrostatic effect for K4 folding and lipid destabilization leading to the microorganisms' death with a high selectivity for the eukaryotic cells at the MIC. Interestingly, the micrographs obtained by electronic microscopy after addition of peptide on bacteria are also consistent with the formation of mixed lipid-peptide objects. Overall, this work supports a detergent-like mechanism for the antimicrobial activity of this peptide.

NMR of molecules interacting with lipids in small unilamellar vesicles.

Detailed characterization of protein, peptide or drug interactions with natural membrane is still a challenge. This review focuses on the use of nuclear magnetic resonance (NMR) for the analysis of interaction of molecules with small unilamellar vesicles (SUV). These phospholipid vesicles are often used as model membranes for fluorescence or circular dichroism experiments. The various NMR approaches for studying molecule-lipid association are reviewed. After a brief survey of the SUV characterization, the use of heteronuclear NMR (phosphorous, carbon, fluorine) is described. Applications of proton NMR through transferred nuclear Overhauser effect to perform structural determination of peptide are presented. Special care is finally given to the influence of the kinetic of the interactions for the proton NMR of bound molecules in SUV, which can constitute a good model for the study of dynamical processes at the membrane surface.

Binding of the dystrophin second repeat to membrane di-oleyl phospholipids is dependent upon lipid packing.

Dystrophin is the genetically deficient protein in Duchenne Muscular Dystrophy. Its C- and N-terminal ends interact with cytoskeletal and membrane proteins, establishing a link between the cytoskeleton and the extracellular matrix. In a previous study, we showed that there is an interaction between the second repeat of the rod domain and membrane phospholipids, which places tryptophan residues in close contact with the membrane. Here, we examine the binding of the dystrophin repeat-2 to small unilamellar vesicles with varying composition. We find that the protein binds predominantly to di-oleyl-phosphatidylserine. The binding as a function of increasing mol% of DOPS appears to be cooperative due to reduction of dimensionality, greatly enhanced in the absence of salts, and partly modulated by pH. Substituting small by large unilamellar vesicles induces a 30-fold lower affinity of the protein for the membrane phospholipids. However, modifying the packing of the acyl chains by introducing lipids such as phosphatidylethanolamine and cholesterol to the vesicle leads to an approximately 7-fold increase in affinity. Taken together, these results show that the binding involves electrostatic forces in addition to hydrophobic ones.