Photogeneration of reactive intermediates

Bond cleavage and formation are key steps in chemistry and biochemistry. The present work investigates the generation of diphenylmethyl cations (Ph2CH+) via photoinduced bond cleavage of diphenylmethyl derivatives with a cationic or neutral leaving group. The resulting Ph2CH+ cations and its numerous derivatives serve as reference electrophiles for one of the most extensive reactivity scales covering 40 orders of magnitude. In chapter 1, the focus is on the initial bond cleavage of diphenylmethyltriphenylphosphonium ions (Ph2CH−PPh3+) exhibiting a cationic leaving group. With the help of state-of-the-art quantum chemical and quantum dynamical methods, the reaction mechanism of the bond cleavage is revealed. Using a reduced model system, the potential energy surfaces can be calculated at the ONIOM level of theory along specially designed reactive coordinates. Two competing reaction channels emerge: a homolytic one in the S1 state and a heterolytic one in the ground state. They are connected via an energetically accessible conical intersection which makes an efficient generation of the observed Ph2CH+ cations feasible. In contradiction with the experiment in polar or moderately polar solvents, quantum dynamical calculations for the isolated molecule reveal the formation of Ph2CH• radicals. While electrostatic solvent effects are negligible in this system, dynamic solvent effects emerge as being essential to explain the molecular mechanism. Two methods with increasing complexity to describe the dynamic impact of the solvent environment are developed. The first approach, the dynamic continuum ansatz, treats the environment implicitly. It uses Stokes’ law and the dynamic viscosity of the solvent in combination with quantum chemically and dynamically evaluated quantities to obtain the decelerating force exerted on the dissociating fragments. The ansatz does not require any fitting of parameters. The second method, the QD/MD approach, is based on an explicit treatment of the solvent surrounding. It combines molecular dynamics (MD) simulations of the reactant in a box of solvent molecules with quantum dynamics (QD) calculations of the reactant’s dynamics. In this way, a more detailed microscopic picture of the molecular process can be derived taking into account individual arrangements of the solvent. Both methods unveil the crucial impact of the solvent cage on the bond cleavage mechanism. It hinders the free dissociation in the S1 state and guides the molecular system to the conical intersection. QD simulations including the non-adiabatic coupling around the conical intersection show the formation of Ph2CH+ within ∼400 fs which compares well with the initial rise of the cation absorption in the experiment. Chapter 2 deals with the position of the counterion X– in the ion pairs Ph2CH−PPh3+ X–, PhCH2−PPh3+ X–, and (p-CF3-C6H4)CH2−PPh3+ X– in solution with X– being Cl–, Br–, BF4–, and SbF6–. These structures are essential to clarify the role of oxidizable counterions like e.g. Cl– during the initial bond cleavage in dichloromethane. The structures determined quantum chemically in dichloromethane show a similar counterion position than in the crystal. They are confirmed by the good accordance of the calculated and measured 1H NMR shifts. The C(α)–H···X– hydrogen bonds account for the pronounced counterion-dependent 1H NMR shifts of the C(α)–H in CD2Cl2. The strong downfield shift of the signals increases according to SbF6– < BF4– << Br– < Cl–. The last part (chapter 3) focuses on the secondary processes within a few picoseconds to several nanoseconds after the C-Cl bond cleavage in diphenylmethylchloride in solution. Initially, the neutral leaving group Cl leads mainly to the formation of radical pairs; only a minor fraction of ion pairs is generated in the beginning. A combined Marcus-Smoluchowski model is used to simulate the interplay between geminate recombination, diffusional separation, and electron transfer of the radical and ion pair populations. The distance-dependent rates of the three processes together with broad distance-dependent population distributions faithfully reproduce the spectroscopically observed dynamics. The majority of Ph2CH+ cations is generated via electron transfer from the radical pairs. The detailed understanding of the secondary processes shows that a high Ph2CH+ cation yield can be expected if the radicals within a pair stay nearby for a long time to achieve an efficient electron transfer and if the resulting ions are separated fast to prevent geminate recombination

The coincidence biosensor tubbyCT reveals local phosphatidylinositol-4,5-bisphosphate synthesis at endoplasmic reticulum-plasma membrane junctions

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays a prominent role in plasma membrane (PM) physiology. It is implicated in the regulation of a variety of cellular functions including exo- and endocytosis, cytoskeleton anchorage, and ion channel activity. Activation of Gq-coupled receptors induces rapid break-down of PI(4,5)P2 by PLCβ. The thereby generated second messengers I(1,4,5)P3 and diacylglycerol (DAG), in turn also stimulate the resynthesis of PI(4,5)P2. Because its precursor phosphytidylinositol (PI) is synthesized in the endoplasmic reticulum (ER), PI transport to the PM is an essential step in replenishment of PI(4,5)P2. This transport has recently been shown to occur by a non-vesicular mechanism at highly specialized contact sites between both membranes, the ER-PM junctions. These membrane contact sites are mediated by membrane tethering proteins, including the Extended Synaptotagmins (E-Syts) and tightened upon intracellular Ca2+ rise, allowing PI transfer to occur. In my work, I discovered the preferential localization of tubbyCT, a known PI(4,5)P2 recognition domain, to E-Syt3-rich ER-PM junctions. Junctional recruitment is mediated by coincidence detection of E-Syt3 and PI(4,5)P2, as shown by co-localization experiments, co-immunoprecipitations and manipulations of PM PI(4,5)P2 content. These dual binding properties allowed, for the first time, the selective investigation of local PI(4,5)P2 dynamics at ER-PM junctions. Using Total Internal Reflection Fluorescence (TIRF) microscopy, TubbyCT revealed the unexpected increase of a local PI(4,5)P2 pool at ER-PM junctions, that was dependent on local synthesis, despite concurrent global PI(4,5)P2 consumption by PLCβ. Pharmacological inhibition of PI(4,5)P2 resynthesis revealed that these local PI(4,5)P2 pool dynamics are required for maintenance and tightening of ER-PM contact sites during PLCβ signaling. Together, my data suggest a model of local metabolic turnover of locally supplied PI, i.e. ‘metabolic channeling’ of PI(4,5)P2 production in the PM. Enrichment at ER-PM contact sites was not restricted to the isolated tubby domain, but was likewise observed with the full-length tubby protein and its close relative TULP3. So far, tubby-like proteins (TULPs) have been implicated in delivery of G protein-coupled receptors to primary cilia. My findings suggest an additional role of TULP proteins at ER-PM junctions not previously recognized

Two cooperative binding sites sensitize PI(4,5)P2 recognition by the tubby domain

Phosphoinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. The dominant PI of the plasma membrane is phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. One of only two PI(4,5)P2 recognition domains characterized in detail is the tubby domain. It is essential for targeting proteins into cilia involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic. Here, we used coarse-grained molecular dynamics simulations to explore PI(4,5)P2 binding by the prototypic tubby domain. The comparatively low PI(4,5)P2 affinity of the previously described canonical binding site is underpinned in a cooperative manner by a previously unknown, adjacent second binding site. Mutations in the previously unknown site impaired PI(4,5)P2-dependent plasma membrane localization in living cells and PI(4,5)P2 interaction in silico, emphasizing its importance for PI(4,5)P2 affinity. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of tubby-like proteins that underlies delivery of ciliary cargo

Photogeneration of reactive intermediates

Bond cleavage and formation are key steps in chemistry and biochemistry. The present work investigates the generation of diphenylmethyl cations (Ph2CH+) via photoinduced bond cleavage of diphenylmethyl derivatives with a cationic or neutral leaving group. The resulting Ph2CH+ cations and its numerous derivatives serve as reference electrophiles for one of the most extensive reactivity scales covering 40 orders of magnitude. In chapter 1, the focus is on the initial bond cleavage of diphenylmethyltriphenylphosphonium ions (Ph2CH−PPh3+) exhibiting a cationic leaving group. With the help of state-of-the-art quantum chemical and quantum dynamical methods, the reaction mechanism of the bond cleavage is revealed. Using a reduced model system, the potential energy surfaces can be calculated at the ONIOM level of theory along specially designed reactive coordinates. Two competing reaction channels emerge: a homolytic one in the S1 state and a heterolytic one in the ground state. They are connected via an energetically accessible conical intersection which makes an efficient generation of the observed Ph2CH+ cations feasible. In contradiction with the experiment in polar or moderately polar solvents, quantum dynamical calculations for the isolated molecule reveal the formation of Ph2CH• radicals. While electrostatic solvent effects are negligible in this system, dynamic solvent effects emerge as being essential to explain the molecular mechanism. Two methods with increasing complexity to describe the dynamic impact of the solvent environment are developed. The first approach, the dynamic continuum ansatz, treats the environment implicitly. It uses Stokes’ law and the dynamic viscosity of the solvent in combination with quantum chemically and dynamically evaluated quantities to obtain the decelerating force exerted on the dissociating fragments. The ansatz does not require any fitting of parameters. The second method, the QD/MD approach, is based on an explicit treatment of the solvent surrounding. It combines molecular dynamics (MD) simulations of the reactant in a box of solvent molecules with quantum dynamics (QD) calculations of the reactant’s dynamics. In this way, a more detailed microscopic picture of the molecular process can be derived taking into account individual arrangements of the solvent. Both methods unveil the crucial impact of the solvent cage on the bond cleavage mechanism. It hinders the free dissociation in the S1 state and guides the molecular system to the conical intersection. QD simulations including the non-adiabatic coupling around the conical intersection show the formation of Ph2CH+ within ∼400 fs which compares well with the initial rise of the cation absorption in the experiment. Chapter 2 deals with the position of the counterion X– in the ion pairs Ph2CH−PPh3+ X–, PhCH2−PPh3+ X–, and (p-CF3-C6H4)CH2−PPh3+ X– in solution with X– being Cl–, Br–, BF4–, and SbF6–. These structures are essential to clarify the role of oxidizable counterions like e.g. Cl– during the initial bond cleavage in dichloromethane. The structures determined quantum chemically in dichloromethane show a similar counterion position than in the crystal. They are confirmed by the good accordance of the calculated and measured 1H NMR shifts. The C(α)–H···X– hydrogen bonds account for the pronounced counterion-dependent 1H NMR shifts of the C(α)–H in CD2Cl2. The strong downfield shift of the signals increases according to SbF6– < BF4– << Br– < Cl–. The last part (chapter 3) focuses on the secondary processes within a few picoseconds to several nanoseconds after the C-Cl bond cleavage in diphenylmethylchloride in solution. Initially, the neutral leaving group Cl leads mainly to the formation of radical pairs; only a minor fraction of ion pairs is generated in the beginning. A combined Marcus-Smoluchowski model is used to simulate the interplay between geminate recombination, diffusional separation, and electron transfer of the radical and ion pair populations. The distance-dependent rates of the three processes together with broad distance-dependent population distributions faithfully reproduce the spectroscopically observed dynamics. The majority of Ph2CH+ cations is generated via electron transfer from the radical pairs. The detailed understanding of the secondary processes shows that a high Ph2CH+ cation yield can be expected if the radicals within a pair stay nearby for a long time to achieve an efficient electron transfer and if the resulting ions are separated fast to prevent geminate recombination

Lipid Fingerprints and Cofactor Dynamics of Light-Harvesting Complex II in Different Membranes

Plant light-harvesting complex II (LHCII) is the key antenna complex for plant photosynthesis. We present coarse-grained molecular dynamics simulations of monomeric and trimeric LHCII in a realistic thylakoid membrane environment based on the Martini force field. The coarse-grained protein model has been optimized with respect to atomistic reference simulations. Our simulations provide detailed insights in the thylakoid lipid fingerprint of LHCII which compares well with experimental data from membrane protein purification. Comparing the monomer and trimeric LHCII reveals a stabilizing effect of trimerization on the chromophores as well as the protein. Moreover, the average chromophore distance shortens in the trimer leading to stronger excitonic couplings. When changing the native thylakoid environment to a model membrane the protein flexibility remains constant, whereas the chromophore flexibility is reduced. Overall, the presented LHCII model lays the foundation to investigate the μs dynamics of this key antenna protein of plants

A locally activatable sensor for robust quantification of organellar glutathione

Glutathione (GSH) is the main determinant of intracellular redox potential and participates in multiple cellular signalling pathways. Achieving a detailed understanding of intracellular GSH homeostasis depends on the development of tools to map GSH compartmentalization and intra-organelle fluctuations. Here we present a GSH-sensing platform for live-cell imaging, termed targetable ratiometric quantitative GSH (TRaQ-G). This chemogenetic sensor possesses a unique reactivity turn-on mechanism, ensuring that the small molecule is only sensitive to GSH in a desired location. Furthermore, TRaQ-G can be fused to a fluorescent protein to give a ratiometric response. Using TRaQ-G fused to a redox-insensitive fluorescent protein, we demonstrate that the nuclear and cytosolic GSH pools are independently regulated during cell proliferation. This sensor was used in combination with a redox-sensitive fluorescent protein to quantify redox potential and GSH concentration simultaneously in the endoplasmic reticulum. Finally, by exchanging the fluorescent protein, we created a near-infrared, targetable and quantitative GSH sensor

Molecular dynamics simulations in photosynthesis

Photosynthesis is regulated by a dynamic interplay between proteins, enzymes, pigments, lipids, and cofactors that takes place on a large spatio-temporal scale. Molecular dynamics (MD) simulations provide a powerful toolkit to investigate dynamical processes in (bio)molecular ensembles from the (sub)picosecond to the (sub)millisecond regime and from the Å to hundreds of nm length scale. Therefore, MD is well suited to address a variety of questions arising in the field of photosynthesis research. In this review, we provide an introduction to the basic concepts of MD simulations, at atomistic and coarse-grained level of resolution. Furthermore, we discuss applications of MD simulations to model photosynthetic systems of different sizes and complexity and their connection to experimental observables. Finally, we provide a brief glance on which methods provide opportunities to capture phenomena beyond the applicability of classical MD

Nogo-a regulates neural precursor migration in the embryonic mouse cortex

Although Nogo-A has been intensively studied for its inhibitory effect on axonal regeneration in the adult central nervous system, little is known about its function during brain development. In the embryonic mouse cortex, Nogo-A is expressed by radial precursor/glial cells and by tangentially migrating as well as postmigratory neurons. We studied radially migrating neuroblasts in wild-type and Nogo-A knockout (KO) mouse embryos. In vitro analysis showed that Nogo-A and its receptor components NgR, Lingo-1, TROY, and p75 are expressed in cells emigrating from embryonic forebrain-derived neurospheres. Live imaging revealed an increased cell motility when Nogo-A was knocked out or blocked with antibodies. Antibodies blocking NgR or Lingo-1 showed the same motility-enhancing effect supporting a direct role of surface Nogo-A on migration. Bromodeoxyuridine (BrdU) labeling of embryonic day (E)15.5 embryos demonstrated that Nogo-A influences the radial migration of neuronal precursors. At E17.5, the normal transient accumulation of radially migrating precursors within the subventricular zone was not detectable in the Nogo-A KO mouse cortex. At E19, migration to the upper cortical layers was disturbed. These findings suggest that Nogo-A and its receptor complex play a role in the interplay of adhesive and repulsive cell interactions in radial migration during cortical developmen

Nogo-A Regulates Neural Precursor Migration in the Embryonic Mouse Cortex

Although Nogo-A has been intensively studied for its inhibitory effect on axonal regeneration in the adult central nervous system, little is known about its function during brain development. In the embryonic mouse cortex, Nogo-A is expressed by radial precursor/glial cells and by tangentially migrating as well as postmigratory neurons. We studied radially migrating neuroblasts in wild-type and Nogo-A knockout (KO) mouse embryos. In vitro analysis showed that Nogo-A and its receptor components NgR, Lingo-1, TROY, and p75 are expressed in cells emigrating from embryonic forebrain–derived neurospheres. Live imaging revealed an increased cell motility when Nogo-A was knocked out or blocked with antibodies. Antibodies blocking NgR or Lingo-1 showed the same motility-enhancing effect supporting a direct role of surface Nogo-A on migration. Bromodeoxyuridine (BrdU) labeling of embryonic day (E)15.5 embryos demonstrated that Nogo-A influences the radial migration of neuronal precursors. At E17.5, the normal transient accumulation of radially migrating precursors within the subventricular zone was not detectable in the Nogo-A KO mouse cortex. At E19, migration to the upper cortical layers was disturbed. These findings suggest that Nogo-A and its receptor complex play a role in the interplay of adhesive and repulsive cell interactions in radial migration during cortical development