Molecular Mechanism for the Dual Alcohol Modulation of Cys-loop Receptors

Cys-loop receptors constitute a superfamily of pentameric ligand-gated ion channels (pLGICs), including receptors for acetylcholine, serotonin, glycine and γ-aminobutyric acid. Several bacterial homologues have been identified that are excellent models for understanding allosteric binding of alcohols and anesthetics in human Cys-loop receptors. Recently, we showed that a single point mutation on a prokaryotic homologue (GLIC) could transform it from a channel weakly potentiated by ethanol into a highly ethanol-sensitive channel. Here, we have employed molecular simulations to study ethanol binding to GLIC, and to elucidate the role of the ethanol-enhancing mutation in GLIC modulation. By performing 1-µs simulations with and without ethanol on wild-type and mutated GLIC, we observed spontaneous binding in both intra-subunit and inter-subunit transmembrane cavities. In contrast to the glycine receptor GlyR, in which we previously observed ethanol binding primarily in an inter-subunit cavity, ethanol primarily occupied an intra-subunit cavity in wild-type GLIC. However, the highly ethanol-sensitive GLIC mutation significantly enhanced ethanol binding in the inter-subunit cavity. These results demonstrate dramatic effects of the F(14′)A mutation on the distribution of ligands, and are consistent with a two-site model of pLGIC inhibition and potentiation.     

High-throughput genotyping of unknown genomic terrain in complex plant genomes: lessons from a case study

Novel high-throughput genotyping technologies have facilitated rapid genotyping of single nucleotide polymorphisms in non-model organisms. Most plant species have complex genomes with a large proportion of their genes having one or more paralogous copies due to single gene duplications and ancient or recent polyploidization events. These paralogous gene copies are potential sources of genotyping errors, and hence genotyping of plant genomes is inherently difficult. Here we present a case study that exemplifies paralog-related problems in high-throughput genotyping of plant genomes. We used the MassARRAY genotyping platform to genotype the LpIRI locus in L. perenne populations; this gene is thought to be involved in low-temperature stress tolerance. The dissection of the molecular genetics underlying the genotyping results provides a good example of how unknown paralogs can mask the true genotype of the locus, instructive to the non-specialist plant researcher and breeder.     

Mismatch between fishway operation and timing of fish movements: a risk for cascading effects in partial migration systems

Habitat fragmentation is a growing problem worldwide. Particularly in river systems, numerous dams and weirs hamper the movement of a wide variety of species. With the aim to preserve connectivity for fish, many barriers in river systems are equipped with fishways (also called fish passages or fish ladders). However, few fishways provide full connectivity. Here we hypothesized that restricted seasonal opening times of fishways can importantly reduce their effectiveness by interfering with the timing of fish migration, for both spring‐ and autumn‐spawning species. We empirically tested our hypothesis, and discuss the possible eco‐evolutionary consequences of affected migration timing. We analyzed movements of two salmonid fishes, spring‐spawning European grayling (Thymallus thymallus) and autumn‐spawning brown trout (Salmo trutta), in Norway's two largest river systems. We compared their timing of upstream passage through four fishways collected over 28 years with the timing of fish movements in unfragmented river sections as monitored by radiotelemetry. Confirming our hypothesis, late opening of fishways delayed the migration of European grayling in spring, and early closure of fishways blocked migration for brown trout on their way to spawning locations during late autumn. We show in a theoretical framework how restricted opening times of fishways can induce shifts from migratory to resident behavior in potamodromous partial migration systems, and propose that this can induce density‐dependent effects among fish accumulating in lower regions of rivers. Hence, fragmentation may not only directly affect the migratory individuals in the population, but may also have effects that cascade downstream and alter circumstances for resident fish. Fishway functionality is inadequate if there is a mismatch between natural fish movements and fishway opening times in the same river system, with ecological and possibly evolutionary consequences for fish populations.     

Changes of the proteinase binding properties and conformation of bovine alpha 2-macroglobulin on cleavage of the thio ester bonds by methylamine

Cleavage of the thio ester bonds of human alpha2-macroglobulin (alpha 2M) by methylamine leads to an extensive conformational change and to inactivation of the inhibitor. In contrast, cleavage of these bonds in bovine alpha 2M only minimally perturbs the hydrodynamic volume of the protein [Dangott, L. J., & Cunningham, L. W. (1982) Biochem. Biophys. Res. Commun. 107, 1243-1251], as well as its spectroscopic properties, as analyzed by ultraviolet difference spectroscopy, circular dichroism, and fluorescence in this work. A conformational change analogous to that undergone by human alpha 2M thus does not occur in the bovine inhibitor. However, changes of several functional properties of bovine alpha 2M are induced by the amine. The apparent stoichiometry of inhibition of trypsin thus is reduced from about 1.2 to about 0.7 mol of enzyme/mol of inhibitor. In spite of this decrease, the interaction with the proteinase induces similar conformational changes in methylamine-treated alpha 2M as in intact alpha 2M, as revealed by spectroscopic analyses, indicating that the mode of binding of the proteinase to the inhibitor is essentially unperturbed by thio ester bond cleavage. The reaction with methylamine also greatly increases the sensitivity of bovine alpha 2M to proteolysis by trypsin at sites other than the "bait" region. Moreover, the second-order rate constant for the reaction with thrombin is reduced by about 10-fold. These results indicate that the thio ester bonds of bovine alpha 2M, although not required per se for the binding of proteinases, nevertheless are responsible for maintaining certain structural features of the inhibitor that are of importance for full activity.     

M?ssbauer, EPR, and magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]1+ cluster with spin S = 3/2

We have studied the Fe protein (Av2) of the Azotobacter vinelandii nitrogenase system with M?ssbauer and EPR spectroscopies and magnetic susceptometry. In the oxidized state the protein exhibits M?ssbauer spectra typical of diamagnetic [4Fe-4S]2+ clusters. Addition of Mg.ATP or Mg.ADP causes a pronounced decline in the quadrupole splitting of the M?ssbauer spectra of the oxidized protein. Our studies show that reduced Av2 in the native state is heterogeneous. Approximately half of the molecules contain a [4Fe-4S]1+ cluster with electronic spin S = 1/2 and half contain a [4Fe-4S]1+ cluster with spin S = 3/2. The former yields the characteristic g = 1.94 EPR signal whereas the latter exhibits signals around g = 5. The magnetization of reduced Av2 is dominated by the spin S = 3/2 form of its [4Fe-4S]1+ clusters. These results explain a long standing puzzle, namely why the integrated spin intensity of the g = 1.94 EPR signal is substantially less than 1 spin/4 Fe atoms. In 50% ethylene glycol, 90% of the clusters are in the spin S = 1/2 form whereas, in 0.4 M urea, 85% are in the S = 3/2 form. In 0.4 M urea, the EPR spectrum of reduced Av2 exhibits well defined resonances at g = 5.8 and 5.15, which we assign to the S = 3/2 system. The EPR and M?ssbauer studies yield a zero-field splitting of 2D approximately equal to -5 cm-1 for this S = 3/2 state.     

Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.

DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.     

M?ssbauer study of CO dehydrogenase from Clostridium thermoaceticum

We have studied with M?ssbauer spectroscopy the metal clusters of CO dehydrogenase from Clostridium thermoaceticum. At potentials greater than -200 mV, all of the approximately 12 irons reside in diamagnetic environments and contribute a quadrupole doublet characteristic of [Fe4S4]2+ clusters. At lower potentials a variety of components are observed. About 40% of the Fe appears to belong to one [Fe4S4]1+ cluster. We have also observed the M?ssbauer spectrum (approximately 18% of Fe) of the complex which yields EPR with g = 2.01, 1.81, and 1.65. Also present is a doublet (9% of Fe) with delta EQ = 2.90 mm/s and delta = 0.70 mm/s, values typical of a ferrous FeS4 complex. This component seems to interact with a nickel site to form an EPR-silent complex with half-integral electronic spin. We have also characterized the iron environments of the S = 1/2 NiFeC complex. This complex contributes approximately 20% of the total M?ssbauer absorption when the EPR signal has approximately 0.35 spins/12 Fe. From isomer shift comparisons in the oxidized and CO-reacted states of this center, we speculate that the NiFeC complex may consist of a nickel site exchange-coupled to a [Fe4S4]2+ cluster. Finally, the M?ssbauer and EPR data, taken together, force us to conclude that current preparations, while homogeneous according to purifications standards, are spectroscopically heterogeneous, thus rendering the development of a model of the cluster types and compositions in this enzyme premature.