RESUMEN
The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de la Membrana/metabolismo , Transporte Biológico , Lipoproteínas/metabolismoRESUMEN
The solubilisation of membrane proteins (MPs) necessitates the overlap of two contradictory events; the extraction of MPs from their native lipid membranes and their subsequent stabilisation in aqueous environments. Whilst the current myriad of membrane mimetic systems provide a range of modus operandi, there are no golden rules for selecting the optimal pipeline for solubilisation of a specific MP hence a miscellaneous approach must be employed balancing both solubilisation efficiency and protein stability. In recent years, numerous diverse lipid membrane mimetic systems have been developed, expanding the pool of available solubilisation strategies. This review provides an overview of recent developments in the membrane mimetic field, with particular emphasis placed upon detergents, polymer-based nanodiscs and amphipols, highlighting the latest reagents to enter the toolbox of MP research.
Asunto(s)
Detergentes/química , Proteínas de la Membrana/química , Micelas , Nanoestructuras/química , SolubilidadRESUMEN
A convenient preparative approach to low-valent phosphorus-rich oligomers is presented. Ligand substitution reactions involving anionic diphosphine ligands of the form [(PR2)2N](-) and [(PPh2)2C5H3](-) and a triphosphenium bromide P(I) precursor result in the formation of phosphorus(I)-containing heterocycles, several of which are of types that have never been prepared before. The methodology described also allows for the preparation of the known heterocycle cyclo-[P(PPh2)N(PPh2)]2 in better yields and purity than the synthetic approach reported previously. Preliminary reactivity studies demonstrate the viability of such zwitterionic oligomers as multidentate ligands for transition metals.
RESUMEN
The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD ß6-ß7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de Escherichia coli , Escherichia coli , Periplasma , Fosfolípidos , Fosfolípidos/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Periplasma/metabolismo , Transporte Biológico , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Modelos Moleculares , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de la MembranaRESUMEN
Attempts to coordinate neutral ligands to low oxidation state indium centers are often hindered by disproportionation pathways that produce elemental indium and higher oxidation state species. In contrast, we find that reactions of the salt, InOTf (OTf=trifluoromethanesulfonate), with α-diimine ligands yielded intensely colored compounds with no evidence of decomposition. X-ray structural analysis of InOTfâ (Mes) DAB(Me) ((Mes) DAB(Me) =N,N-dimesityl-2,3-dimethyl-diazabutadiene; 1) reveals a discrete molecular compound with a pyramidal coordination environment at the indium center, consistent with the presence of a stereochemically active lone pair of electrons on indium and a neutral diazabutadiene chelate ligand. The use of the less-electron-rich (Mes) DAB(H) ligand ((Mes) DAB(H) =N,N-dimesityl-diazabutadiene) engenders dramatically different reactivity and produces a metallopolymer (InOTfâ (Mes) DAB(H) )∞ (2) linked via CC and InIn bonds. The difference in reactivity is rationalized by cyclic voltammetry and DFT studies that suggest more facile electron transfer from In(I) to the (Mes) DAB(H) and bis(aryl)acenaphthenequinonediimine (BIAN) ligands. Solution EPR spectroscopy indicates the presence of non-interacting ligand-based radicals in solution, whereas solid-state EPR studies reflect the presence of a thermally accessible spin triplet consistent with reversible CC bond cleavage.
RESUMEN
The outer membrane of Gram-negative bacteria provides a formidable barrier, essential for both pathogenesis and antimicrobial resistance. Biogenesis of the outer membrane requires the transport of phospholipids across the cell envelope. Recently, YhdP was implicated as a major protagonist in the transport of phospholipids from the inner membrane to the outer membrane however the molecular mechanism of YhdP mediated transport remains elusive. Here, utilising AlphaFold, we observe YhdP to form an elongated assembly of 60 ß strands that curve to form a continuous hydrophobic groove. This architecture is consistent with our negative stain electron microscopy data which reveals YhdP to be approximately 250 Å in length and thus sufficient to span the bacterial cell envelope. Furthermore, molecular dynamics simulations and in vivo bacterial growth assays indicate essential helical regions at the N- and C-termini of YhdP, that may embed into the inner and outer membranes respectively, reinforcing its envelope spanning nature. Our in vivo crosslinking data reveal phosphate-containing substrates captured along the length of the YhdP groove, providing direct evidence that YhdP transports phospholipids. This finding is congruent with our molecular dynamics simulations which demonstrate the propensity for inner membrane lipids to spontaneously enter the groove of YhdP. Collectively, our results support a model in which YhdP bridges the cell envelope, providing a hydrophobic environment for the transport of phospholipids to the outer membrane.
RESUMEN
A series of tin(II) triflate and chloride salts in which the cations are complexed by either cyclic or acyclic polyether ligands and which have well-characterized single-crystal X-ray structures are investigated using a variety of experimental and computational techniques. Mössbauer spectroscopy illustrates that the triflate salts tend to have valence electrons with higher s-character, and solid-state NMR spectroscopy reveals marked differences between superficially similar triflate and chloride salts. Cyclic voltammetry investigations of the triflate salts corroborate the results of the Mössbauer and NMR spectroscopy and reveal substantial steric and electronic effects for the different polyether ligands. MP2 and DFT calculations provide insight into the effects of ligands and substituents on the stability and reactivity of the low-valent metal atom. Overall, the investigations reveal the existence of more substantial binding between tin and chlorine in comparison to the triflate substituent and provide a rationale for the considerably increased reactivity of the chloride salts.
RESUMEN
The use of the univalent indium reagent [In([18]crown-6)][OTf] as a donor is investigated by its reactions with acceptors including InX(3) (X=Cl, Br, I). The donor-acceptor complexes of the form [X([18]crown-6)In-InX(3)] obtained in this manner represent the first new isomeric form of indium(II) halides identified for at least five decades. The formation of such complexes appears to be particularly favorable and they are isolated as products in many reactions involving low-valent indium, a halide source, and [18]crown-6. A convenient solution-phase synthesis of In[ECl(4)] salts is reported. This facile and direct syntheses of In[ECl(4)] (E=Al, Ga, In) salts allows for the in situ preparation and isolation of crown-ether complexes of the form [In([18]crown-6)][ECl(4)], whose existence had been postulated but never confirmed. Solution-phase and solid-state NMR experiments reveal that these compounds can exist as either donor-acceptor complexes or ionic salts, depending on the phase of the system, the nature of the solvent employed, and the identity of the metalate anion involved. Similar investigations into the effect of a smaller crown ether allow for the isolations of salts containing the cation [In([15]crown-5)](+). Computational investigations into the nature of the crowned univalent indium donor fragments, and on the donor-acceptor complexes produced, demonstrate the influence of anionic substituents on the reactivity of lone pair of electrons of the In(I) center. Natural bond orbital (NBO) analysis of donor-acceptor models shows that the composition of the E-E bond MO should provide the ability to predict which models should form stable complexes.
RESUMEN
In the title compound, [In(CH(3))(2)(C(12)H(24)O(6))](CF(3)O(3)S), two of the In-O distances within the cation are significantly shorter than the other four. The In(III) atom is in a distorted hexa-gonal-bipyramidal coordination geometry in which the C-In-C angle is 175.44â (12)°. The crystal structure is stabilized by weak inter-molecular C-Hâ¯O hydrogen bonds.
RESUMEN
The alkaloid (-)-securinine was synthesized in 18 steps and 16% overall yield from trans-4-hydroxy-l-proline.
Asunto(s)
Azepinas/síntesis química , Lactonas/síntesis química , Piperidinas/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de Anillo en Puente , EstereoisomerismoRESUMEN
A significant amount of gas production has been observed in the bromate-pyrocatechol oscillator under high concentrations of bromate and pyrocatechol. The observation is in contrast to the general perception that aromatic compounds can form bromate-based oscillators that are free of gas bubbles, which is a desired property in investigating pattern formation. Analysis with (1)H NMR, (13)C NMR, mass spectrometry, and X-ray crystallography illustrate the production of 5-(dibromomethylene)-2(5H)-furanone from pyrocatechol, where the loss of one carbon atom from the aromatic ring causes the formation of gas bubbles. Possible mechanisms have been proposed to explain the observed phenomenon.
Asunto(s)
4-Butirolactona/análogos & derivados , Bromatos/química , Catecoles/química , Gases/síntesis química , 4-Butirolactona/síntesis química , 4-Butirolactona/química , Cristalografía por Rayos X , Gases/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Estructura MolecularRESUMEN
Fit for a king: Cationic complexes of Ge(II) can be prepared by using crown ethers to stabilize and protect the germanium center. Three different crown ethers were employed: [12]crown-4 (see structure, Ge teal, O red, C gray), [15]crown-5, and [18]crown-6. The structures of the cationic complexes depend on the cavity size of the crown ether and on the substituent on germanium.
RESUMEN
Solid-state (35)Cl NMR (SSNMR) spectroscopy is shown to be a useful probe of structure and polymorphism in HCl pharmaceuticals, which constitute ca. 50% of known pharmaceutical salts. Chlorine NMR spectra, single-crystal and powder X-ray diffraction data, and complementary ab initio calculations are presented for a series of HCl local anesthetic (LA) pharmaceuticals and some of their polymorphs. (35)Cl MAS SSNMR spectra acquired at 21.1 T and spectra of stationary samples at 9.4 and 21.1 T allow for extraction of chlorine electric field gradient (EFG) and chemical shift (CS) parameters. The sensitivity of the (35)Cl EFG and CS tensors to subtle changes in the chlorine environments is reflected in the (35)Cl SSNMR powder patterns. The (35)Cl SSNMR spectra are shown to serve as a rapid fingerprint for identifying and distinguishing polymorphs, as well as a useful tool for structural interpretation. First principles calculations of (35)Cl EFG and CS tensor parameters are in good agreement with the experimental values. The sensitivity of the chlorine NMR interaction tensor parameters to the chlorine chemical environment and the potential for modeling these sites with ab initio calculations hold much promise for application to polymorph screening for a wide variety of HCl pharmaceuticals.
Asunto(s)
Cloruros/química , Ácido Clorhídrico/química , Espectroscopía de Resonancia Magnética/métodos , Preparaciones Farmacéuticas/química , Polímeros/química , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética/normas , Modelos Moleculares , Estructura Molecular , Estándares de ReferenciaRESUMEN
The metathesis reaction of InCl3 with Me2NCH2CH2SNa or the redox reaction of indium metal with elemental iodine and the disulfide (Me2NCH2CH2S)2 yield the indium bis(thiolate) complexes (Me2NCH2CH2S)2InX [X = Cl (3) and I (4)], respectively. Compounds 3 and 4 may be further reacted with the appropriate sodium thiolate salts to afford the heteroleptic tris(thiolate) complexes (Me2NCH2CH2S)2InSR [R = 4-MeC6H4 (5), 4-MeOC6H4 (6), and Pr (7)]. Reaction of 2,6-Me2C6H3SNa with 4 affords (Me2NCH2CH2S)2InS(2,6-Me2C6H3) (8), while no reaction is observed with 3, suggesting a greater reactivity for 4. All isolated compounds were characterized by elemental analysis, melting point, and Fourier transform IR and 1H and 13C{1H} NMR spectroscopies. X-ray crystallographic analyses of 3-6 show a bicyclic arrangement and a distorted trigonal-bipyramidal geometry for In in all cases. The two sulfur and one halogen (3 and 4) or three sulfur (5 and 6) atoms occupy equatorial positions, while the nitrogen atoms of the chelating (dimethylamino)ethanethiolate ligands occupy the axial positions. The metric parameters of the (Me2NCH2CH2S)2In framework were found to change minimally upon variation of the X/SR ligand, while the solubility of the corresponding compounds in organic solvents varied greatly. 1H NMR studies in D2O showed that 6 and 7 react slowly with an excess of the tripeptide l-glutathione and that the rate of reaction is affected by the pendant thiolate ligand -SR.