A role for taurine in mitochondrial function

The mitochondrial pH gradient across the inner-membrane is stabilised by buffering of the matrix. A low-molecular mass buffer compound has to be localised in the matrix to maintain its alkaline pH value. Taurine is found ubiquitously in animal cells with concentrations in the millimolar range and its pKa value is determined to 9.0 (25°C) and 8.6 (37°C), respectively. Localisation of such a low-molecular buffer in the mitochondrial matrix, transforms the matrix into a biochemical reaction chamber for the important matrix-localised enzyme systems. Three acyl-CoA dehydrogenase enzymes, which are pivotal for beta-oxidation of fatty acids, are demonstrated to have optimal activity in a taurine buffer. By application of the model presented, taurine depletion caused by hyperglycemia could provide a link between mitochondrial dysfunction and diabetes

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires measuring spatiotemporally precise neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires measuring spatiotemporally precise neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators

Molecular Adaptation of Photoprotection: Triplet States in Light-Harvesting Proteins

The photosynthetic light-harvesting systems of purple bacteria and plants both utilize specific carotenoids as quenchers of the harmful (bacterio)chlorophyll triplet states via triplet-triplet energy transfer. Here, we explore how the binding of carotenoids to the different types of light-harvesting proteins found in plants and purple bacteria provides adaptation in this vital photoprotective function. We show that the creation of the carotenoid triplet states in the light-harvesting complexes may occur without detectable conformational changes, in contrast to that found for carotenoids in solution. However, in plant light-harvesting complexes, the triplet wavefunction is shared between the carotenoids and their adjacent chlorophylls. This is not observed for the antenna proteins of purple bacteria, where the triplet is virtually fully located on the carotenoid molecule. These results explain the faster triplet-triplet transfer times in plant light-harvesting complexes. We show that this molecular mechanism, which spreads the location of the triplet wavefunction through the pigments of plant light-harvesting complexes, results in the absence of any detectable chlorophyll triplet in these complexes upon excitation, and we propose that it emerged as a photoprotective adaptation during the evolution of oxygenic photosynthesis

Acyl-CoA Dehydrogenasen: Mechanistische Untersuchungen mit der "Medium chain"-Acyl-CoA-Dehydrogenase

Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the a,b-dehydrogenation of fatty acid acyl-CoA thioesters. Medium chain acyl-CoA dehydrogenase (MCAD) is one of the best-studied members of this family. The a,b-dehydrogenation reaction involves the concerted C-H bonds cleavage of the substrate. First, the active site base, Glu376-COO-, removes a proton by and then a hydride is transferred to the flavin N(5) position of FAD. In my thesis MCAD several mechanistic details of the dehydrogenation reaction for MCAD were investigated. For this, among other things, a mutant of MCAD was created, which carries a C-terminal "His Tag". Addition of affinity His Tag facilitates purification of recombinant MCAD. For the investigation of the mechanism above several E376- or/and E99-MCAD mutants were used. Last one received an earlier attention since the Glu99 is located underneath of the active site of MCAD. This residue affects ionizations inside the active center cavity. Many studies were focused on E376Q-MCAD mutant. This mutant was highly inactive, because the glutamine does not play the role of the base. However its residual activity is 1/100000 of that of wtMCAD. This is a small value, but has the same order of magnitude as those found in non-catalyzed reactions. Proton inventory technique was suitable for mechanistic study of this mutant. Apart from this, it was observed that the log of rates of dehydrogenation increases linearly with the pH suggesting HO- as a reactant. A similar dependence was observed with Glu376Gln+Glu99Gly-MCAD. Thus, activity and reduction studies exclude Glu99 as a candidate for proton abstraction in the first step of dehydrogenation. E376Q-MCAD mutant reflected a large unexpected solvent isotope effect of approx. 8.5. The large isotope effects resulted from proton inventory experiments are attributed to the change in state of several H-bonds that occur during the process. A further investigation concerns the role of a special H-bond between N(5) of the flavin cofactor and Thr168-OH. However, an amino acid functional group that forms such a H-bond is strictly conserved in the ACAD familily (Thr or Ser). In the absence of this H-bond (T168A-MCAD) two effects could be observed: a) electronic influence on the substrate activation as well as on the redox potential of the flavin; b) steric - this H-bond is involved in the fine-tuning of the orientation of the flavin cofactor and ligand. Another threonine residue (Thr136) modulates the redox potential of the flavin (approx. -30 mV compared to wtMCAD 1.4 Kcal M-1). Thus e.g. with the Thr136Ala mutant the cofactor was partially reduced by the substrate, which is attributed to decrease of the redox potential. These experiments were supported by theoretical calculations, which were accomplished by Olga Dmitrenko working at Univ. of Delaware (USA) in Prof. R. Bach group

Development of Polyurethane/Peptide-Based Carriers with Self-Healing Properties

In situ-forming gels with self-assembling and self-healing properties are materials of high interest for various biomedical applications, especially for drug delivery systems and tissue regeneration. The main goal of this research was the development of an innovative gel carrier based on dynamic inter- and intramolecular interactions between amphiphilic polyurethane and peptide structures. The polyurethane architecture was adapted to achieve the desired amphiphilicity for self-assembly into an aqueous solution and to facilitate an array of connections with peptides through physical interactions, such as hydrophobic interactions, dipole-dipole, electrostatic, π–π stacking, or hydrogen bonds. The mechanism of the gelation process and the macromolecular conformation in water were evaluated with DLS, ATR-FTIR, and rheological measurements at room and body temperatures. The DLS measurements revealed a bimodal distribution of small (~30–40 nm) and large (~300–400 nm) hydrodynamic diameters of micelles/aggregates at 25 °C for all samples. The increase in the peptide content led to a monomodal distribution of the peaks at 37 °C (~25 nm for the sample with the highest content of peptide). The sol–gel transition occurs very quickly for all samples (within 20–30 s), but the equilibrium state of the gel structure is reached after 1 h in absence of peptide and required more time as the content of peptide increases. Moreover, this system presented self-healing properties, as was revealed by rheological measurements. In the presence of peptide, the structure recovery after each cycle of deformation is a time-dependent process, the recovery is complete after about 300 s. Thus, the addition of the peptide enhanced the polymer chain entanglement through intermolecular interactions, leading to the preparation of a well-defined gel carrier. Undoubtedly, this type of polyurethane/peptide-based carrier, displaying a sol–gel transition at a biologically relevant temperature and enhanced viscoelastic properties, is of great interest in the development of medical devices for minimally invasive procedures or precision medicine

Solvent Isotope Effects in Reactions of Human Medium-Chain Acyl-CoA Dehydrogenase Active Site Mutants

Glu376, the base involved in substrate αH+ abstraction at the active center of medium-chain acyl-CoA dehydrogenase (MCAD), has been mutated to Gln and Gly. The mutants are active; however, their rates of dehydrogenation are lowered by approximately 5 orders of magnitude. Binding of the substrate octanoyl-CoA to Glu376Gln-MCAD involves (at least) two steps. The ensuing dehydrogenation reaction that corresponds to reduction of the flavin cofactor also occurs in two phases. These are interpreted to consist of a first, reversible step, followed by a slower, practically irreversible one. For Glu376Gln-MCAD, the log of the rates of dehydrogenation increases linearly with pH (slope = 1) in the pH range of 6-10, suggesting HO- as a reactant. The rates of the same reactions in D2O have the same pD profile and reflect a solvent kinetic isotope effect (SKIE) of ≈8.5. Glu376Gln+Glu99Gly-MCAD (studied to assess the role of Glu99 also present at the bottom of the active center cavity) has activities and activity profiles similar to those of Glu376Gln-MCAD. This excludes Glu99 as the active center base for Glu376Gln-MCAD catalysis. Proton inventories for the two phases of the dehydrogenation reaction were investigated at 4 and 25°C. The inventories at 25°C reflect a SKIE of ≈4.5; the profiles are "bowl-shaped", in which a transition-state contribution predominates. The profiles for the 4°C reaction are very unusual. That for the first phase can be analyzed on a two-step model with one step (80% rate-limiting) having a conformational reorganization with an isotope effect of 90-100, from small isotope effects at many protein sites, and the other step (20% rate-limiting) having an inverse isotope effect of ca. 2, characteristic of the reaction of hydroxide ion as a base. For the second phase, only a contribution from many protein sites with a KIE of ≈4.5 is observed. The results are compatible with a very rigid active site framework that must undergo rearrangements for dehydrogenation to take place, and specifically to allow access of HO-, the reactant that must neutralize the H+ abstracted from the αC-H substrate. The large isotope effects are attributed to the changes in state of several H-bonds that occur during the process

Temperature Induced Gelation and Antimicrobial Properties of Pluronic F127 Based Systems

Different formulations containing Pluronic F127 and polysaccharides (chitosan, sodium alginate, gellan gum, and κ-carrageenan) were investigated as potential injectable gels that behave as free-flowing liquid with reduced viscosity at low temperatures and displayed solid-like properties at 37 °C. In addition, ZnO nanoparticles, lysozyme, or curcumin were added for testing the antimicrobial properties of the thermal-sensitive gels. Rheological investigations evidenced small changes in transition temperature and kinetics of gelation at 37 °C in presence of polysaccharides. However, the gel formation is very delayed in the presence of curcumin. The antimicrobial properties of Pluronic F127 gels are very modest even by adding chitosan, lysozyme, or ZnO nanoparticles. A remarkable enhancement of antimicrobial activity was observed in the presence of curcumin. Chitosan addition to Pluronic/curcumin systems improves their viscoelasticity, antimicrobial activity, and stability in time. The balance between viscoelastic and antimicrobial characteristics needs to be considered in the formulation of Pluronic F127 gels suitable for biomedical and pharmaceutical applications