Elucidation of molecular kinetic schemes from macroscopic traces using system identification

Overall cellular responses to biologically-relevant stimuli are mediated by networks of simpler lower-level processes. Although information about some of these processes can now be obtained by visualizing and recording events at the molecular level, this is still possible only in especially favorable cases. Therefore the development of methods to extract the dynamics and relationships between the different lower-level (microscopic) processes from the overall (macroscopic) response remains a crucial challenge in the understanding of many aspects of physiology. Here we have devised a hybrid computational-analytical method to accomplish this task, the SYStems-based MOLecular kinetic scheme Extractor (SYSMOLE). SYSMOLE utilizes system-identification input-output analysis to obtain a transfer function between the stimulus and the overall cellular response in the Laplace-transformed domain. It then derives a Markov-chain state molecular kinetic scheme uniquely associated with the transfer function by means of a classification procedure and an analytical step that imposes general biological constraints. We first tested SYSMOLE with synthetic data and evaluated its performance in terms of its rate of convergence to the correct molecular kinetic scheme and its robustness to noise. We then examined its performance on real experimental traces by analyzing macroscopic calcium-current traces elicited by membrane depolarization. SYSMOLE derived the correct, previously known molecular kinetic scheme describing the activation and inactivation of the underlying calcium channels and correctly identified the accepted mechanism of action of nifedipine, a calcium-channel blocker clinically used in patients with cardiovascular disease. Finally, we applied SYSMOLE to study the pharmacology of a new class of glutamate antipsychotic drugs and their crosstalk mechanism through a heteromeric complex of G protein-coupled receptors. Our results indicate that our methodology can be successfully applied to accurately derive molecular kinetic schemes from experimental macroscopic traces, and we anticipate that it may be useful in the study of a wide variety of biological systems

Stereodynamics of E/Z isomerization in rotaxanes through mechanical shuttling and covalent bond rotation

We report on a set of rotaxanes with symmetrical axles equipped with a central amide group that installs E/Z stereoisomerism owing to the ring position along the axle. Isomerization by concomitant rotation about the amide bond and ring shuttling along the axle was thoroughly characterized in different solvents. The results trigger a discussion on core concepts, such as microscopic reversibility and transition state theory, and provide insights for designing molecules capable to transform and transmit motion between subcomponents

Roles of Serine 101, Histidine 310 and Valine 464 in the Reaction Catalyzed by Choline Oxidase from Arthrobacter Globiformis

The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions, as such it is of potential interest for the genetic engineering of crops that do not naturally possess efficient pathways for the synthesis of glycine betaine, and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, one of the best characterized enzymes belonging to this pathway is the flavin-dependent choline oxidase from Arthrobacter globiformis. In this enzyme, choline oxidation proceeds through two reductive half-reactions and two oxidative half-reactions. In each of the reductive half-reactions the FAD cofactor is reduced to the anionic hydroquinone form (2 e- reduced) which is followed by an oxidative half-reaction where the reduced FAD cofactor is reoxidized by molecular oxygen with formation and release of hydrogen peroxide. In this dissertation the roles of selected residues, namely histidine at position 310, valine at position 464 and serine at position 101, that do not directly participate in catalysis in the reaction catalyzed by choline oxidase have been elucidated. The effects on the overall reaction kinetics of these residues in the protein matrix were investigated by a combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects as well as X-ray crystallography. A comparison of the kinetic data obtained for the variant enzymes to previous data obtained for wild-type choline oxidase are consistent with the valine residue at position 464 being important for the oxidative half-reaction as well as the positioning of the catalytic groups in the active site of the enzyme. The kinetic data obtained for the serine at position 101 shows that serine 101 is important for both the reductive and oxidative half-reactions. Finally, the kinetic data for histidine at position 310 suggest that this residue is essential for both the reductive and oxidative half-reactions

Synthesis and investigation of clusters of like-charged ions in OH-functionalized ionic liquids

In dieser Arbeit untersuchten wir systematisch die Bildung von kationischen (c-c)-Clustern in OH-funktionalisierten ionischen Flüssigkeiten (ILs) mit einer Vielzahl von experimentellen und theoretischen Methoden. Der Synthese der ILs folgte die umfassende Untersuchung der (c-c)-Cluster und deren Einflussfaktoren in der Gasphase, der kondensierten Phase und an der Fest/Flüssig-Grenzfläche. Mit IR-Spektroskopie, Neutronenbeugung und quantenchemischen Methoden konnten wir zeigen, dass die kooperativen Wasserstoffbrücken in (c-c)-Clustern stärker sind als die zwischen Kation und Anion.In this work, we systematically investigated the formation of cationic clusters in OH-functionalised ionic liquids (ILs) using a plethora of experimental and theoretical methods. The synthesis and characterization of well-suited sets of ILs was followed by a comprehensive study of cationic clusters and their influencing factors in the gas phase, the condensed phase and at the solid/liquid interface. Infrared spectroscopy, neutron diffraction and quantum chemical methods showed that cooperative hydrogen bonding in cationic clusters is counterintuitively stronger than between cation and anion

High temperature sensitivity is intrinsic to voltage-gated potassium channels.

Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. Here we report that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors

Atomic Scale Investigation of Pressure Induced Phase Transitions in the solid State

In this work, atomic scale investigation of pressure-induced transformations in the solid state have been carried out. A series of compounds including GaN, ZnO, CaF2, and AgI, in addition to elemental phosphorus have been studied. The corresponding transition mechanisms have been elucidated with a clear description of atomic displacements and intermediate structures involved therein. In the first group of compounds, the long standing debate on the transition path of the wurtzite(WZ)-to-rocksalt(RS) transition in semiconductors, GaN and ZnO was resolved using geometrical modeling combined with molecular dynamics (MD) simulations conducted in the frame of transition path sampling (TPS) method. In GaN, a two-step mechanism through a metastable intermediate phase with a tetragonal structure iT has been revealed from simulations. In ZnO, the tetragonal intermediate structure was kinetically less stable, although still part of the real transition mechanism. It appeared at the interface between WZ and RS as consequence of a layers shearing. The transition regime in ZnO was characterized by a competition between iT structure and another hexagonal intermediate with hexagonal symmetry iH. Although possible, the latter is not functional for the transition. In both cases, GaN and ZnO, two points of agreement with experiments have been revealed. The tilting of structures after transition, and the phonon mode softening associated with atomic displacements leading to the tetragonal structure iT In the second group of compounds, the investigation of transitions in superionic conductors, CaF2 and AgI, demonstrated a different and particular behavior of atomic motion under pressure. The solid-solid reconstruction of CaF2 structure was shown to be initiated and precedented by high disorder of the anionic sublattice. The percolation of fluoride ions through voids in the fluorite structure created a thin interface of liquid like state. The sparce regions caused by the departure of anions facilitates the cation sublattice reconstruction. In AgI, ion diffusion during the wurtzite/zincnlende(ZB)$rocksalt transition was more pronounced due to the extended stacking disorder WZ/ZB. The Ag+ ions profited not only from the structure of the interface but used the combination of interstitial voids offered by both phases, WZ and ZB, to achieve long diffusion paths and cause the cation sublattice to melt. Clearly, a proper account for such phenomena cannot be provided by geometry-designed mechanisms based on symmetry arguments. In phosphorus, the question of how the stereochemically active lone pairs are reorganized during the orthorhombic (PI) to trigonal (PV) structural transition was answered by means of simulations. Computation was performed at different levels theory. First, the mechanism of the transition was obtained from TPS MD simulations. MD runs were performed within density functional tight binding method (DFTB). The analysis of atomic displacements along the real transformation path indicated a fast bond switching mechanism. In a second step, the nature of the interplay between orbitals of phosphorus during the bond switching was investigated. A simultaneous deformation of lone pair and P−P bond showed a mutual switching of roles during the transformation. This interplay caused a low dimensional polymerization of phosphorus under pressure. The corresponding structure formed as zigzag linear chain of fourfold coordinated phosphorus atoms (· · ·(P(P2))n · · ·) at the interface between PI and PV phases. A further result of this work was the development of a simulation strategy to incorporate defects and chemical doping to structural transformations. On top of the transition path sampling iterations, a Monte Carlo like procedure is added to stepwise substitute atoms in the transforming system. Introducing a chemically different dopant to a pure system represents a perturbation to the energy landscape where the walk between different phases is performed. Therefore, any change in the transition regime reflects the kinetic preference of a given structural motif at times of phase formation. This method was applied to the elucidation of WZ-RS transition mechanism in the series of semiconducting compounds AlN, GaN, and InN. Simulations showed that In atoms adopt the same transformation mechanism as in GaN and favor it, while Al atoms demonstrated a significant reluctance to the path going through tetragonal intermediate iT. The difference between transition regime in mixed systems InxGa1−xN and AlxGa1−xN is in agreement with experiments on high pressure behavior of AlN, GaN, and InN. While transitions in GaN and InN are reversible down to ambient conditions, AlN is stable. The work presented in this thesis constitutes the seed of new perspectives in the understanding of pressure-induced phase transformations in the solid state, where the physics and the chemistry are brought together by means of computer simulations

Proceedings of the Thirteenth International Conference on Time-Resolved Vibrational Spectroscopy

The thirteenth meeting in a long-standing series of “Time-Resolved Vibrational Spectroscopy” (TRVS) conferences was held May 19th to 25th at the Kardinal Döpfner Haus in Freising, Germany, organized by the two Munich Universities - Ludwig-Maximilians-Universität and Technische Universität München. This international conference continues the illustrious tradition of the original in 1982, which took place in Lake Placid, NY. The series of meetings was initiated by leading, world-renowned experts in the field of ultrafast laser spectroscopy, and is still guided by its founder, Prof. George Atkinson (University of Arizona and Science and Technology Advisor to the Secretary of State). In its current format, the conference contributes to traditional areas of time resolved vibrational spectroscopies including infrared, Raman and related laser methods. It combines them with the most recent developments to gain new information for research and novel technical applications. The scientific program addressed basic science, applied research and advancing novel commercial applications. The thirteenth conference on Time Resolved Vibrational Spectroscopy promoted science in the areas of physics, chemistry and biology with a strong focus on biochemistry and material science. Vibrational spectra are molecule- and bond-specific. Thus, time-resolved vibrational studies provide detailed structural and kinetic information about primary dynamical processes on the picometer length scale. From this perspective, the goal of achieving a complete understanding of complex chemical and physical processes on the molecular level is well pursued by the recent progress in experimental and theoretical vibrational studies. These proceedings collect research papers presented at the TRVS XIII in Freising, German

The persistence and genetic support of genes encoding antibiotic resistance in oral bacteria.

Antibiotic-resistant bacteria present a serious public health threat and studies investigating the prevalence of antibiotic-resistant bacteria and the genes encoding for antibiotic resistance (GEAR) are essential. Such studies help us to understand how antibiotic-resistant bacteria and GEAR are obtained, transferred and maintained within a population. The aim of this study was to identify the prevalence and maintenance of antibiotic-resistant bacteria in the oral microbiota of children aged 4-6 years who had not previously taken antibiotics. Bacteria resistant to penicillin, ampicillin, tetracycline and erythromycin were widespread among the children studied. Tetracycline-resistant bacteria were found in 98% of the children sampled and the tetracycline resistance determinants most responsible for this resistance were tet(M) and tet(W). The tetracycline-resistant bacteria were maintained within 15 children over a period of twelve months. In three of these children the tet(M) gene was found to persist in a variety of genera and this was found to be contained within a Tn9/6-like element by Southern blot analysis. The tetracycline resistance determinant tet(S) was found for the first time in Streptococcus intermedius . It was found to be transferable to Enterococcus faecalis and other Streptococcus spp. by filter matings. The tet(S) gene was shown to be present on a novel Tn9/f5-like element designated Tn97r5S. The similarities between Tn916 and Tn976S included the presence of conjugative and excision and integration modules. However the tet(M) in Tn976 had been completely replaced by tet(S) in Tn9/f5S. The gene tet(32) was also found for the first time in Streptococcus parasanguinis and Eubacterium saburreum, previously it had been found in a human colonic bacterium K10. The upstream region of the oral tet(32) gene in E. saburreum 41.2T.2 was found to be more closely related to upstream sequences of tet(W) in Roseburia sp. A2-123, upstream sequences of tet(W) within TnB1230 (originally isolated from Butyrivibrio fibrisolvens ) and orf!4, orf25 and orf26 from Tn5397. This work provides further evidence that antibiotic-resistant bacteria are prevalent in the oral microbiota of children and that the genes responsible, especially those encoding resistance to tetracycline, can be maintained in the oral microbiota even when the children have not been directly exposed to antibiotics. These findings show that tetracycline resistance determinants can be mosaic in structure and can be easily transferred due to their containment within conjugative transposons. Such elements then undergo evolutionary changes, whereby recombination of the conjugative transposon modules result in new genetic elements

Elucidation of the Mechanisms of Nucleosome Binding and Repositioning by a Chromatin Remodeler: Monomeric ISWI Remodels Nucleosomes Through a Random Walk

The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The goal of this work was to characterize quantitatively the nucleosome repositioning activity, and associated processes of nucleotide binding, DNA binding, and nucleosome binding, of the chromatin remodeler ISWI. ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. The necessary first step in determining how these essential enzymes catalyze the repositioning of nucleosomes is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. Toward this goal we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon ISWI binding to these substrates. We determined that one ISWI molecule binds to a 20 bp double stranded DNA substrate with an affinity of (18 ± 2) nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants 1/&beta1 = (1.3 ± 0.6) nM and 1/&beta2 = (13 ± 7) nM2. Furthermore, in order to better understand the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogs on substrate binding by ISWI. While the affinity of ISWI to binding nucleosome substrate with short lengths of flanking DNA was not affected by presence of nucleotides, the affinity of ISWI for binding DNA substrate is weakened in the presence of non-hydrolysable ATP analogs but not in the presence of ADP. Additionally, using standard electrophoresis assays we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing different lengths of DNA symmetrically flanking an initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a novel spectrophotometric assay for nucleosome repositioning we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed under various ISWI concentrations revealed that monomeric ISWI is sufficient to account for the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning suggesting that DNA translocation by ISWI is not energetically rate limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction. In conclusion, the characterization of the mechanism of nucleosome binding and repositioning by the chromatin remodeler ISWI presented in this dissertation provides a foundation for future studies aiming to understand how various regulatory elements influence the function of ISWI