Recombinant expression, synthesis, purification, and solution structure of arenicin

Arenicins are 21-residue cationic antimicrobial peptides, isolated from marine polychaeta Arenicola marina. In order to determine a high-resolution three-dimensional structure of arenicin-2, the recombinant peptide was overexpressed as a fused form in Escherichia coli. Both arenicin isoforms were synthesized using the Fmoc-based solid-phase strategy. Recombinant and synthetic arenicins were purified, and their antimicrobial and spectroscopic properties were analyzed. NMR investigation shows that in water solution arenicin-2 displays a prolonged beta-hairpin, formed by two antiparallel beta-strands and stabilized by one disulfide and nine hydrogen bonds. A significant right-handed twist in the beta-sheet is deprived the peptide surface of amphipathicity. CD spectroscopic analysis indicates that arenicin-2 binds to the SDS and DPC micelles, and conformation of the peptide is significantly changed upon binding. Arenicin strongly binds to anionic lipid (POPE/POPG) vesicles in contrast with zwitterionic (POPC) ones. These results suggest that arenicins are membrane active peptides and point to possible mechanism of their selectivity toward bacterial cells.     

Tyk2 mediates effects of urokinase on human vascular smooth muscle cell growth

The urokinase (uPA)/uPA receptor (uPAR) system plays a role in the response of the vessel wall to injury, presumably by modulating vascular smooth muscle cell (VSMC) functional behaviour. The Jak/Stat signaling pathway has been implicated to mediate the uPA/uPAR-directed cell migration and proliferation in VSMC. We have therefore investigated the underlying molecular mechanisms, which remained not completely understood. In particular, we aimed at identification of the kinase involved in the signaling cascade leading to Stat1 phosphorylation by uPA and its impact on VSMC growth. We performed expression in VSMC of kinase-deficient mutant forms of the Janus kinases Jak1 and Tyk2 and used different cell culture models imitating the response to vascular injury. We provide evidence that Tyk2, but not Jak1, mediates uPA-induced Stat1 phosphorylation and VSMC growth inhibition and suggest a novel function for Tyk2 as an important modulator of the uPA-directed VSMC functional behaviour at the place of injury.     

Crystal structures of the carboxyl terminal domain of rat 10-formyltetrahydrofolate dehydrogenase: implications for the catalytic mechanism of aldehyde dehydrogenases

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes an NADP+-dependent dehydrogenase reaction resulting in conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. This reaction is a result of the concerted action of two catalytic domains of FDH, the amino-terminal hydrolase domain and the carboxyl-terminal aldehyde dehydrogenase domain. In addition to participation in the overall FDH mechanism, the C-terminal domain is capable of NADP+-dependent oxidation of short chain aldehydes to their corresponding acids. We have determined the crystal structure of the C-terminal domain of FDH and its complexes with oxidized and reduced forms of NADP. Compared to other members of the ALDH family, FDH demonstrates a new mode of binding of the 2'-phosphate group of NADP via a water-mediated contact with Gln600 that may contribute to the specificity of the enzyme for NADP over NAD. The structures also suggest how Glu673 can act as a general base in both acylation and deacylation steps of the reaction. In the apo structure, the general base Glu673 is positioned optimally for proton abstraction from the sulfur atom of Cys707. Upon binding of NADP+, the side chain of Glu673 is displaced from the active site by the nicotinamide ring and contacts a chain of highly ordered water molecules that may represent a pathway for translocation of the abstracted proton from Glu673 to the solvent. When reduced, the nicotinamide ring of NADP is displaced from the active site, restoring the contact between Cys707 and Glu673 and allowing the latter to activate the hydrolytic water molecule in deacylation.     

Nitration of tryptophan 372 in succinyl-CoA:3-ketoacid CoA transferase during aging in rat heart mitochondria

The main objective of this study was to test the hypothesis that in vivo post-translational modifications in proteins, induced by the endogenously generated reactive oxygen and nitrogen molecules, can alter protein function and thereby have an effect on metabolic pathways during the aging process. Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT), the mitochondrial enzyme involved in the breakdown of ketone bodies in the extrahepatic tissues, was identified in rat heart to undergo age-associated increase in a novel, nitro-hydroxy, addition to tryptophan 372, located in close proximity ( approximately 10 A) of the enzyme active site. Between 4 and 24 months of age, the molar content of nitration was more than doubled while specific enzyme activity increased significantly. The amount of SCOT protein, however, remained unchanged. In vitro treatment of heart mitochondrial soluble proteins with relatively low concentrations of peroxynitrite enhanced the nitration as well as specific activity of SCOT. Results of this study identify tryptophan to be a specific target of nitration in vivo, for the first time. We hypothesize that increases in tryptophan nitration of SCOT and catalytic activity constitute a plausible mechanism for the age-related metabolic shift toward enhanced ketone body consumption as an alternative source of energy supply in the heart.     

Mechanisms of discrimination between cobalamins and their natural analogues during their binding to the specific B12-transporting proteins

Three proteins, intrinsic factor (IF), transcobalamin (TC), and haptocorrin (HC), all have an extremely high affinity for the cobalamins (Cbls, Kd approximately 5 fM) but discriminate these physiological ligands from Cbl analogues with different efficiencies decreasing in the following order: IF > TC > HC. We investigated interactions of these proteins with a number of ligands: Cbl, fluorescent conjugate CBC, two base-off analogues [pseudo-coenzyme B12 (pB) and adenosyl factor A (fA)], and a baseless corrinoid cobinamide. Protein-ligand encounter and the following internal rearrangements in both molecules were registered as a change in the fluorescence of CBC (alone or mixed with other ligands), a transition in absorbance of pB and fA (base-off --> on-base conversion), and alterations in the molecular mass of two split IF domains. The greater complexity of the binding kinetics followed better Cbl specificity (HC < TC < IF). On the basis of the experimental results, we propose a general binding model with three major steps: (1) initial attachment of the ligand to the high-affinity C-domain, (2) primary assembly of N- and C-domains, and (3) slow adjustments and fixation of the ligand at the domain-domain interface. Since step 3 was characteristic of highly specific TC and especially IF, we suggest its particular importance for ligand recognition. The designed models revealed the absolute Kd values for a group of analogues. Calculations show that most of them could potentially bind to the specific transporters IF and TC under physiological conditions. Implications of this finding and the protective role of HC are discussed.     

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus

The kinetics of the oxidation of fully-reduced ba(3) cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa(3)-type oxidases. However the F intermediate in ba(3) oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P-->F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F-->O transition in ba(3) oxidase is close to that observed during the F-->O transition in the aa(3) oxidases. However, the P(R)-->F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.     

Correlation of electron transfer rate in photosynthetic reaction centers with intraprotein dielectric properties

A number of the electrogenic reactions in photosystem I, photosystem II, and bacterial reaction centers (RC) were comparatively analyzed, and the variation of the dielectric permittivity (epsilon) in the vicinity of electron carriers along the membrane normal was calculated. The value of epsilon was minimal at the core of the complexes and gradually increased towards the periphery. We found that the rate of electron transfer (ET) correlated with the value of the dielectric permittivity: the fastest primary ET reactions occur in the low-polarity core of the complexes within the picosecond time range, whereas slower secondary reactions take place at the high-polarity periphery of the complexes within micro- to millisecond time range. The observed correlation was quantitatively interpreted in the framework of the Marcus theory. We calculated the reorganization energy of ET carriers using their van der Waals volumes and experimentally determined epsilon values. The electronic coupling was calculated by the empirical Moser-Dutton rule for the distance-dependent electron tunneling rate in nonadiabatic ET reactions. We concluded that the local dielectric permittivity inferred from the electrometric measurements could be quantitatively used to estimate the rate constant of ET reactions in membrane proteins with resolved atomic structure with the accuracy of less than one order of magnitude.     

Genetic differentiation by sexual conflict

Sexual conflict has been suggested as a general cause of genetic diversification in reproductive characters, and as a possible cause of speciation. We use individual-based simulations to study the dynamics of sexual conflict in an isolated diploid population with no spatial structure. To explore the effects of genetic details, we consider two different types of interlocus interaction between female and male traits, and three different types of intra-locus interaction. In the simulations, sexual conflict resulted in at least the following five regimes: (1) continuous coevolutionary chase, (2) evolution toward an equilibrium, (3) cyclic coevolution, (4) extensive genetic differentiation in female traits/genes only, and (5) extensive genetic differentiation in both male and female traits/genes. Genetic differentiation was hardly observed when the traits involved in reproduction were determined additively and interacted in a trait-by-trait way. When the traits interacted in a component-by-component way, genetic differentiation was frequently observed under relatively broad conditions. The likelihood of genetic differentiation largely depended on the number of loci and the type of within-locus dominance. With multiple loci per trait, genetic differentiation was often observed but sympatric speciation was typically hindered by recombination. Sympatric speciation was possible but only under restrictive conditions. Our simulations also highlight the importance of stochastic effects in the dynamics of sexual conflict.     

Synthesis of a quaternary polyprenyl ammonium salt

Reaction of primary C(55)-allylic alcohol moraprenol (WT(3)C(7-9)-OH, a polyprenol from mulberry leaves) with triethylamine in the presence of phosphorus oxychloride leads to a quaternary ammonium chloride with a good yield (72%) and high cis-stereoselectivity of the terminal isoprene unit. Cationic polyprenyl derivatives may be useful for transfection and immunological studies.     

5(6)-carboxyfluorescein revisited: new protecting group, separation of isomers, and their spectral properties on oligonucleotides

Pentafluorophenyl esters of 5- and 6-carboxyfluorescein-3',6'-O-dipivalate can be easily separated in multigram quantities by column chromatography. The individual isomers were converted into stable phosphoramidites suitable for oligonucleotide synthesis. The use of the cyclohexylcarbonyl (Chc) protecting group instead of pivaloyl (Piv) facilitates the separation of isomers. The fluorescence spectra of 5- and 6-carboxyfluoresceins on oligonucleotides were compared.