Seasonal variation of high-molecular-weight compounds in the water-soluble fraction of organic urban aerosols

Aerosol samples were collected in Zurich, Switzerland, at an urban background site and were analyzed with size exclusion chromatography (SEC) and laser/desorption ionization mass spectrometry (LDI-MS) for water-soluble organic compounds with high molecular weight. Daily samples were collected during two campaigns in winter and summer, for 1 month each. The concentration of high-molecular-weight compounds (humic-like substances (HULIS)) was between 0.4 and 4 μg/m3 in winter and summer. The most intense signals in the LDI-MS mass spectra were measured between m/z150 and 500, comparing well with the mode of the two main high mass peaks determined with SEC corresponding to masses between 200 and 600 Da. For the maximum molecular weight, however, different results were obtained by the two techniques: whereas a maximum molecular weight between 1300 and 3300 Da was found with SEC, hardly any peaks above m/z700 were measured with LDI-MS. During summer the maximum molecular weight of HULIS (determined with SEC) correlates positively with several parameters such as ozone and increased temperature indicative of enhanced atmospheric photo-oxidation. The HULIS concentration also correlates positively with the oxalic acid concentration in the particles. This suggests that HULIS are generated by secondary processes in summer. The lack of such correlations during winter suggests that other sources and processes might be important during colder seasons.

Mass spectrometric study of molecular and ionic sublimation of lanthanum triiodide

The molecular and ionic composition of saturated vapor over lanthanum triiodide was studied by Knudsen effusion mass spectrometry. The (LaI3)n molecules (n = 1–3) and the [I(LaI3)n]− ions (n = 0–4) were observed. The partial pressures of the molecules were determined and the enthalpies of sublimation, ΔsH° (298.15 K) in kJ mol−1, in the form of monomers (304 ± 7), dimers (428 ± 25), and trimers (455 ± 50) were obtained by the second and third laws of thermodynamics. The enthalpy of formation, ΔfH° (298.15 K) in kJ mol−1, of the LaI3 (−376 ± 10), La2I6 (−932 ± 25), La3I9 (−1585 ± 50) molecules and the LaI4− (−841 ± 24), La2I7− (−1486 ± 32) ions were determined. The electron work function, φe = 3.5 ± 0.3 eV, for the LaI3 crystal was calculated from the thermochemical cycle.

TimX, a novel player in protein import across the inner mitochondrial membrane of T. brucei

Mitochondrial protein import is an essential function of the unique mitochondrion in T. brucei as roughly 1000 different nuclear encoded proteins need to be correctly localized to their mitochondrial subcompartment. For this reason the responsible import machinery is expected to be similarly complex as in other Eukaryotes. This was recently demonstrated for the translocation machinery in the outer mitochondrial membrane. In contrast, the composition of the inner membrane import machinery and the exact molecular pathway(s) taken by various substrates are still ill-defined. To elucidate this further, we performed a pulldown analysis of epitope tagged TbTim17 in combination with quantitative mass spectrometry. By this we identified novel components of the mitochondrial import machinery in trypanosomes. One of these, TimX, is an essential mitochondrial membrane protein of 42 kDa that is unique to kinetoplastids. This protein migrates on Blue Native PAGE in a high molecular weight complex similar to TbTim17. Ablation of either of the two proteins leads to a destabilization of the complex containing the other protein. Furthermore, its involvement in protein import could be demonstrated by in vivo and in vitro protein import assays. This corroborates that TimX together with TbTim17 forms a protein import complex in the inner mitochondrial membrane. As TbTim17 the TimX protein was subjected to pulldown analysis in combination with quantitative mass spectrometry. The overlap of candidates defined by these two sets of IPs likely defines further components of the inner membrane translocase which are presently being analyzed. In summary our study on novel components of the trypanosome mitochondrial protein import system gives us fascinating new insights into evolution of the mitochondrion.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is often considered as one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Its protein import systems are therefore of great interest. Recently, it was shown that the outer mitochondrial membrane protein translocase is of similar complexity yet different composition than in other eukaryotes (1). In the inner membrane however, only a single orthologue of the pore forming Tim17/22/23 protein family was identified and termed TbTim17. Based on this finding it has been suggested that, instead of separate TIM22 and TIM23 complexes as in other eukaryotes, trypanosomes may have a single multifunctional translocase of the inner mitochondrial membrane (TIM) of reduced complexity. To elucidate the composition of the trypanosomal TIM complex we performed co-immunoprecipitations (CoIP) of epitope-tagged TbTim17 in combination with SILAC-based quantitative mass spectrometry. This led to the identification of 22 highly enriched TbTim17-interacting proteins. We tagged two of the top-scoring proteins for reciprocal CoIP analyses and recovered a set of ten proteins that are highly enriched in all three CoIPs. These proteins are excellent candidates for core subunits of the trypanosomal TIM complex. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a novel trypanosomatid-specific 42 kDa protein, termed Tim42, and two putative orthologues of probably inactive rhomboid proteases were chosen for further analysis. All three proteins are essential in both life cycle stages and in a cell line that can grow in the absence of mitochondrial DNA. Additionally, their ablation by RNAi results in a strong protein import defect both in vivo and in vitro. Blue native PAGE reveals that Tim42, like TbTim17 is present in a high molecular weight complex. Moreover, ablation of either Tim42 or TbTim17 leads to a destabilization of the complex containing the other protein, suggesting a tight interaction of the two proteins. In summary our study shows that unlike anticipated trypanosomes have a highly complex TIM translocase that has extensively been redesigned. We have characterized three novel TIM subunits that have never been associated with mitochondrial protein import before. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. Our study provides insight into mitochondrial evolution over large phylogenetic distances and suggests an exciting analogy between protein translocation systems of mitochondria and the ER.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Effect of lipid bilayer properties on the photocycle of green proteorhodopsin

The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-β-d-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.

Excitonic splittings in molecular dimers: why static ab initio calculations cannot match them

After decades of research on molecular excitons, only few molecular dimers are available on which exciton and vibronic coupling theories can be rigorously tested. In centrosymmetric H-bonded dimers consisting of identical (hetero)aromatic chromophores, the monomer electronic transition dipole moment vectors subtract or add, yielding S0 → S1 and S0 → S2 transitions that are symmetry-forbidden or -allowed, respectively. Symmetry breaking by 12C/13C or H/D isotopic substitution renders the forbidden transition weakly allowed. The excitonic coupling (Davydov splitting) can then be measured between the S0 → S1 and S0 → S2 vibrationless bands. We discuss the mass-specific excitonic spectra of five H-bonded dimers that are supersonically cooled to a few K and investigated using two-color resonant two-photon ionization spectroscopy. The excitonic splittings Δcalc predicted by ab initio methods are 5–25 times larger than the experimental excitonic splittings Δexp. The purely electronic ab initio splittings need to be reduced (“quenched”), reflecting the coupling of the electronic transition to the optically active vibrations of the monomers. The so-called quenching factors Γ < 1 can be determined from experiment (Γexp) and/or calculation (Γcalc). The vibronically quenched splittings Γ·Δcalc are found to nicely reproduce the experimental exciton splittings.

Kinetoplastid-specific pATOM36 mediates membrane insertion of a subset of mitochondrial outer membrane proteins

In trypanosomes, as in other eukaryotes, more than 95% of all mitochondrial proteins are imported into the mitochondrion. The recently characterized multisubunit ATOM complex mediates import of essentially all proteins across the outer mitochondrial membrane in T. brucei. Moreover, an additional protein termed pATOM36, which is loosely associated with the ATOM complex, has been implicated in the import of only a subset of mitochondrial matrix proteins. Here we have investigated more precisely which role pATOM36 plays in mitochondrial protein import. RNAi mediated ablation of pATOM36 specifically depletes a subset of ATOM complex subunits and as a consequence results in the collapse of the ATOM complex as shown by Blue native PAGE. In addition, a SILAC-based global proteomic analysis of uninduced and induced pATOM36 RNAi cells together with in vitro import experiments suggest that pATOM36 might be a novel protein insertase acting on a subset of alpha-helically anchored mitochondrial outer membrane proteins. Identification of pATOM36 interaction partners by co-immunoprecipitation together with immunofluorescence analysis furthermore shows that unexpectedly a fraction of the protein is associated with the tripartite attachment complex (TAC). This complex is essential for proper inheritance of the kDNA as it forms a physical connection between the kDNA and the basal body of the flagellum throughout the cell cycle. Thus, the presence of pATOM36 in the TAC provides an exciting link between mitochondrial protein import and kDNA inheritance.

Materials properties from electron density distributions: Molecular magnetism and linear optical properties

The general goal of this thesis is correlating observable properties of organic and metal-organic materials with their ground-state electron density distribution. In a long-term view, we expect to develop empirical or semi-empirical approaches to predict materials properties from the electron density of their building blocks, thus allowing to rationally engineering molecular materials from their constituent subunits, such as their functional groups. In particular, we have focused on linear optical properties of naturally occurring amino acids and their organic and metal-organic derivatives, and on magnetic properties of metal-organic frameworks. For analysing the optical properties and the magnetic behaviour of the molecular or sub-molecular building blocks in materials, we mostly used the more traditional QTAIM partitioning scheme of the molecular or crystalline electron densities, however, we have also investigated a new approach, namely, X-ray Constrained Extremely Localized Molecular Orbitals (XC-ELMO), that can be used in future to extracted the electron densities of crystal subunits. With the purpose of rationally engineering linear optical materials, we have calculated atomic and functional group polarizabilities of amino acid molecules, their hydrogen-bonded aggregates and their metal-organic frameworks. This has enabled the identification of the most efficient functional groups, able to build-up larger electric susceptibilities in crystals, as well as the quantification of the role played by intermolecular interactions and coordinative bonds on modifying the polarizability of the isolated building blocks. Furthermore, we analysed the dependence of the polarizabilities on the one-electron basis set and the many-electron Hamiltonian. This is useful for selecting the most efficient level of theory to estimate susceptibilities of molecular-based materials. With the purpose of rationally design molecular magnetic materials, we have investigated the electron density distributions and the magnetism of two copper(II) pyrazine nitrate metal-organic polymers. High-resolution X-ray diffraction and DFT calculations were used to characterize the magnetic exchange pathways and to establish relationships between the electron densities and the exchange-coupling constants. Moreover, molecular orbital and spin-density analyses were employed to understand the role of different magnetic exchange mechanisms in determining the bulk magnetic behaviour of these materials. As anticipated, we have finally investigated a modified version of the X-ray constrained wavefunction technique, XC-ELMOs, that is not only a useful tool for determination and analysis of experimental electron densities, but also enables one to derive transferable molecular orbitals strictly localized on atoms, bonds or functional groups. In future, we expect to use XC-ELMOs to predict materials properties of large systems, currently challenging to calculate from first-principles, such as macromolecules or polymers. Here, we point out advantages, needs and pitfalls of the technique. This work fulfils, at least partially, the prerequisites to understand materials properties of organic and metal-organic materials from the perspective of the electron density distribution of their building blocks. Empirical or semi-empirical evaluation of optical or magnetic properties from a preconceived assembling of building blocks could be extremely important for rationally design new materials, a field where accurate but expensive first-principles calculations are generally not used. This research could impact the community in the fields of crystal engineering, supramolecular chemistry and, of course, electron density analysis.

Molecular-Based Magnetism in Bimetallic Two-Dimensional Oxalate-Bridged Networks. An X-ray and Neutron Diffraction Study

Bimetallic, oxalate-bridged compounds with bi- and trivalent transition metals comprise a class of layered materials which express a large variety in their molecular-based magnetic behavior. Because of this, the availability of the corresponding single-crystal structural data is essential to the successful interpretation of the experimental magnetic results. We report in this paper the crystal structure and magnetic properties of the ferromagnetic compound {[N(n-C3H7)4][MnIICrIII(C2O4)3]}n (1), the crystal structure of the antiferromagnetic compound {[N(n-C4H9)4][MnIIFeIII(C2O4)3]}n (2), and the results of a neutron diffraction study of a polycrystalline sample of the ferromagnetic compound {[P(C6D5)4][MnIICrIII(C2O4)3]}n (3). Crystal data:  1, rhombohedral, R3c, a = 9.363(3) Å, c = 49.207(27) Å, Z = 6; 2, hexagonal, P63, a = 9.482(2) Å, c = 17.827(8) Å, Z = 2. The structures consist of anionic, two-dimensional, honeycomb networks formed by the oxalate-bridged metal ions, interleaved by the templating cations. Single-crystal field dependent magnetization measurements as well as elastic neutron scattering experiments on the manganese(II)−chromium(III) samples show the existence of long-range ferromagnetic ordering behavior below Tc = 6 K. The magnetic structure corresponds to an alignment of the spins perpendicular to the network layers. In contrast, the manganese(II)−iron(III) compound expresses a two-dimensional antiferromagnetic ordering.