Overexpression and purification of the primase domain of bacteriophage P4 gp alpha for structure determination by NMR

Bacteriophage P4 is a temperate phage of E. coli and other Gram-negative prokaryotes. Its alpha gene encodes a 777-residue multifunctional protein, gpa, that is essential and sufficient for the initiation of P4 DNA replication. The gpa protein attaches to the P4 origin of replication, unwinds the DNA, and synthesizes a short strand of RNA which acts as a primer for the DNA polymerase of the host organism. These three activities, which in bacteria are carried out by three individual proteins, can be attributed to distinct gpa domains. The N-terminus of gpa constitutes the RNA polymerase, or primase, domain. We have overexpressed the gpa[1-373] primase domain in E. coli on minimal medium and are currently optimizing the purification procedure. It is intended to obtain the structure in solution of uniformly 13C,15N-labeled gpa[1-373] by ultra high-field 3D NMR spectroscopy. Primases of eubacteria and their phages and plasmids share sequence similarities, and comparison of a bacteriophage primase structure with the known structures of crystals of E. coli DnaG and P. furiosus primase domains could yield valuable insights into the mechanism of DNA-directed RNA polymerase activity

TheαM1 transmembrane segment of the nicotinic acetylcholine receptor interacts strongly with model membranes

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) plays a role in the regulation of the activity of this important ligand-gated ion channel. The lipid composition of the host membrane affects conformational equilibria of the nAChR and several classes of inhibitors, most notably anaesthetics, interact directly or indirectly with the four transmembrane M-segments, M1–M4, of the nAChR subunits. It has proven difficult to gain insight into structure–function relationships of the M-segments in the context of the entire receptor and the biomembrane environment. However, model membrane systems are well suited to obtain detailed information about protein–lipid interactions. In this solid-state NMR study, we characterized interactions between a synthetic αM1 segment of the T. californica nAChR and model membranes of different phosphatidylcholine (PC) lipids. The results indicate that αM1 interacts strongly with PC bilayers: the peptide orders the lipid acyl chains and induces the formation of small vesicles, possibly through modification of the lateral pressure profile in the bilayer. The multilamellar vesicle morphology was stabilized by the presence of cholesterol, implying that either the rigidity or the bilayer thickness is a relevant parameter for αM1–membrane interactions, which also has been suggested for the entire nAChR. Our results suggest that the model systems are to a certain extent sensitive to peptide–bilayer hydrophobic matching requirements, but that the lipid response to hydrophobic mismatch alone is not the explanation. The effect of αM1 on different PC bilayers may indicate that the peptide is conformationally flexible, which in turn would support a membrane-mediated modulation of the conformation of transmembrane segments of the nAChR

Lipid bilayer functionalization of multiwalled carbon nanotubes

Integration of the technologically favorable mechanical and electrical properties of carbon nanotubes (CNTs) with the specific recognition properties of proteins could enable the development of novel bioelectronic, in particular biosensing, applications. The hydrophobic graphene surface of CNTs, however, is not a biological substrate and as-synthesized CNTs aggregate in aqueous solution. CNTs can be easily dispersed by non-covalent binding of surfactants like sodium dodecyl sulfate, but the use of such detergents is undesirable because they unfold proteins and degrade cell membranes. We show here that carbon nanotubes can also be dispersed by coating them with biocompatible surfactant analogs. Incubation of multiwalled CNTs with sonicated vesicles of synthetic phospholipids resulted in a stable aqueous suspension of the nanotubes, also after removal of the vesicles by centrifugation. When the vesicles were doped with a fluorescently labelled lipid, the washed CNTs could be observed by fluorescence microscopy. Additionally, atomic force microscopy indicated that the nanotubes were coated by a smooth layer, with occasional defects or transitions to a second layer. These discontinuities were consistently 4-5 nm deep, the typical thickness of a lipid bilayer. It can thus be concluded that vesicle fusion results in the formation of lipid bilayers on the surface of multiwalled CNTs. We addressed the influence of vesicle size, lipid acyl chain saturation, lipid head group charge, CNT surface modification, and CNT diameter on the efficiency of lipid coating. Significantly, it proved possible to include a fluorescently labelled transmembrane peptide in nanotube-supported bilayers, and we are currently investigating whether this can also be achieved for membrane protein

The Nachbac Pore: creation and characterisation of a KcsA-like sodium channel

Voltage-gated sodium channels (VGSC) are integral membrane proteins responsible for the transient flux of sodium ions across cell membranes in response to changes in membrane potential. In humans as well as lower eukaryotes they are essential for homeostasis and normal functioning, and mutations in them are associated with a range of disease states. Although potassium channels, which are members of the same large family of voltage-gated channels have been well characterized, much less known about the structural features of sodium channels. For potassium ion channels, an important advance in understanding resulted from the determination of the three dimensional structure of the bacterial potassium channel KcsA, a simplified channel composed only of two transmembrane segments per subunit present in the tetrameric structure. In 2001, Ren et al found that bacteria also possess simplified versions of sodium channels, although in this case the individual subunits of all the homologues that have been identified thus far possess six transmembrane segments, which include both a pore-forming subdomain (S5-S6) and a voltage-sensing subdomain (S1-S4). Here we report on the creation of a smaller KcsA-like pore-only version of a sodium channel from the B. halodurans VGSC (pNaChBac), engineered to contain S5-S6 plus the C-terminal region of the NaChBac channel. The NaChBac pore has been expressed and purified from E. coli membranes, solubilised in detergent micelles, reconstituted into lipid vesicles and characterized for its secondary structure and thermal stability, as well as its electrophysiological properties from single-channel recordings, providing new insight into features of sodium channel structure and function

Catalisadores de níquel (II) contendo ligantes imina-furano aplicados a dimerização seletiva do etileno

Neste trabalho, uma nova classe de catalisadores de NiII contendo ligantes imina-furano tais como NiCl2{N-((5-metilfurano-2-il)metileno)-2-fenoxietanimina} (1), NiCl2{N-((5- metilfurano-2-il)metileno)-2-fenoxibenzimina} (2), NiCl2 {2-metoxifenil-N-((5-metilfurano-2- il)metileno) metanimina} (3), NiCl2{N-((furano-2-il)metileno)-2-fenoxibenzimina} (4) foram sintetizados e caracterizados por espectrometria de massas de alta resolução e espectroscopia na região do infra-vermelho. Estes catalisadores de níquel, quando ativados com metilaluminoxano (MAO), apresentaram freqüências de rotação (FRs) entre 14.700 e 206.100(mol C2H4)×(mol Ni−1 h−1) com boa seletividade para produção de buteno-1 (63,2 – 83,2%). O complexo 2, na presença de MAO, apresentou maior FR com relação a atividades catalíticas apresentadas pelos complexos 1, 3 e 4 este resultado pode estar associado a maior rigidez do ligante imina-furano, que confere ao catalisador mais estabilidade. Considerando a maior atividade de 2, o mesmo foi utilizado em reações de otimização, nas quais foram avaliados os efeitos das condições reacionais como solvente, razão molar [Al/Ni], tipo/procedência de cocatalisador, quantidade de catalisador e temperatura) sobre a FR e seletividade. Este estudo mostrou que estas variáveis exercem forte influência sobre a FR e a seletividade do sistema, principalmente no que tange ao tipo de cocatalisador empregado nas reações de oligomerização. Neste caso, o emprego de sesquicloreto de etilalumínio (Et3Al2Cl3, EASC) produz um sistema catalítico mais ativo que 2/MAO [FR = 206.100 vs. 57.300 (mol C2H4)×(mol Ni−1 h−1)]. Por outro lado, o uso deste cocatalisador ocasiona uma drástica redução na seletividade para buteno-1, chegando apenas a 11,9 % e associado a produção de uma grande quantidade de butenos internos (88,1 %) e hexenos (12,3 %). Sob condições otimizadas ([Ni] = 10μmol, 50°C, tempo = 20 min, 20 bar de etileno, [Al/Ni] = 500), precatalisador 2 apresentou uma FR = 56.100 (mol C2H4)×(mol Ni−1 h−1) e seletividade de 82,0% para produção de buteno-1.In this work, a set of four new NiII catalysts based on imine-furane ligands such as NiCl2{N- ((5-metilfurano-2-il)metileno)-2-fenoxietanimine} (1), NiCl2{N-((5-metilfurano-2-il)metileno)- 2-fenoxibenzimine} (2), NiCl2 {2-metoxifenil-N-((5-metilfurano-2-il)metileno) metanimine} (3), NiCl2{N-((furano-2-il)metileno)-2-fenoxibenzimine} (4) was synthetized and characterized by high-resolution mass spectra and infrared spectroscopy. All nickel precatalysts, activated with methylaluminoxane (MAO), exhibited high activities for ethylene oligomerization [TOF = 14,700 – 57,300 mol(ethylene)(mol(Ni))−1 h−1)] with good selectivities for 1-butene produced (63.2 – 83.2%). The catalytic performance was substantially affected by the ligand environment regarding the imine pendant group, and the substituents on the furfural group. For this case, the precatalyst 2 showed higher activity related to those presented by precatalysts 1, 3 and 4. Based on these preliminary results, precatalyst 2 was selected for further optimization, investigating the influence of temperature, oligomerization time, ethylene pressure, [Al/Ni] ratio, amount of catalyst, and cocatalyst type. This study showed that these parameters has strongly influence on TOF and selectivity. For instance, the activation of nickel precatalyst 2 with ethylaluminum sesquichloride (Et3Al2Cl3, EASC) instead of MAO produced a significantly better catalyst system than 2/MAO (TOF = 206,100 vs. 57,300 (mol C2H4)×(mol Ni−1 h−1); however, the 1- butene selectivity was drastically reduced, attaining only 11.8% with a concomitant production of larger amounts of internal butenes (88.1%) and hexenes (12.3%). Under optimized conditions ([Ni] = 10μmol, 50°C, oligomerization time = 20 min, 20 bar ethylene, [Al/Ni] = 500), precatalyst 2 led to TOF = 56,100 (mol C2H4)×(mol Ni−1 h−1) and 82.0% selectivity for 1- butene

The αM1 segment of the nicotinic acetylcholine receptor exhibits conformational flexibility in a membrane environment

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) is predominantly α-helical, and of the four distinctly different transmembrane M-segments, only the helicity of M1 is ambiguous. In this study, we have investigated the conformation of a membrane-embedded synthetic M1 segment by solid-state nuclear magnetic resonance (NMR) methods. A 35-residue peptide representing the extended αM1 domain 206–240 of the Torpedo californica nAChR was synthesized with specific 13C- and 15N-labelled amino acids, and was incorporated in different phosphatidylcholine model membranes. The chemical shift of the isotopic labels was resolved by magic angle spinning (MAS) NMR and could be related to the secondary structure of the αM1 analog at the labelled sites. Our results show that the membrane-embedded αM1 segment forms an unstable α-helix, particularly near residue Leu18 (αLeu223 in the entire nAChR). This non-helical tendency was most pronounced when the peptide was incorporated in fully hydrated phospholipid bilayers, with an estimated 40–50% of the peptides having an extended conformation at position Leu18. We propose that the conserved proline residue at position 16 in the αM1 analog imparts a conformational flexibility on the M1 segments that could enable membrane-mediated modulation of nAChR activity

Sensitivity of single membrane-spanning alpha-helical peptides to hydrophobic mismatch with a lipid bilayer: Effects on backbone structure, orientation, and extent of membrane incorporation

The extent of matching of membrane hydrophobic thickness with the hydrophobic length of transmembrane protein segments potentially constitutes a major director of membrane organization. Therefore, the extent of mismatch that can be compensated, and the types of membrane rearrangements that result, can provide valuable insight into membrane functionality. In the present study, a large family of synthetic peptides and lipids is used to investigate a range of mismatch situations. Peptide conformation, orientation, and extent of incorporation are assessed by infrared spectroscopy, tryptophan fluorescence, circular dichroism, and sucrose gradient centrifugation. It is shown that peptide backbone structure is not significantly affected by mismatch, even when the extent of mismatch is large. Instead, this study demonstrates that for tryptophan-flanked peptides the dominant response of a membrane to large mismatch is that the extent of incorporation is reduced, when the peptide is both too short and too long. With increasing mismatch, a smaller fraction of peptide is incorporated into the lipid bilayer, and a larger fraction is present in extramembranous aggregates. Relatively long peptides that remain incorporated in the bilayer have a small tilt angle with respect to the membrane normal. The observed effects depend on the nature of the flanking residues: long tryptophan-flanked peptides do not associate well with thin bilayers, while equisized lysine-flanked peptides associate completely, thus supporting the notion that tryptophan and lysine interact differently with membrane-water interfaces. The different properties that aromatic and charged flanking residues impart on transmembrane protein segments are discussed in relation to protein incorporation in biological systems