Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.     

Interaction of transducin with uncoordinated 119 protein (UNC119): implications for the model of transducin trafficking in rod photoreceptors

The key visual G protein, transducin undergoes bi-directional translocations between the outer segment (OS) and inner compartments of rod photoreceptors in a light-dependent manner thereby contributing to adaptation and neuroprotection of rods. A mammalian uncoordinated 119 protein (UNC119), also known as Retina Gene 4 protein (RG4), has been recently implicated in transducin transport to the OS in the dark through its interaction with the N-acylated GTP-bound transducin-α subunit (Gα(t1)). Here, we demonstrate that the interaction of human UNC119 (HRG4) with transducin is dependent on the N-acylation, but does not require the GTP-bound form of Gα(t1). The lipid specificity of UNC119 is unique: UNC119 bound the myristoylated N terminus of Gα(t1) with much higher affinity than a prenylated substrate, whereas the homologous prenyl-binding protein PrBP/δ did not interact with the myristoylated peptide. UNC119 was capable of interacting with Gα(t1)GDP as well as with heterotrimeric transducin (G(t)). This interaction of UNC119 with G(t) led to displacement of Gβ(1)γ(1) from the heterotrimer. Furthermore, UNC119 facilitated solubilization of G(t) from dark-adapted rod OS membranes. Consistent with these observations, UNC119 inhibited rhodopsin-dependent activation of G(t), but had no effect on the GTP-hydrolysis by Gα(t1). A model for the role of UNC119 in the IS→OS translocation of G(t) is proposed based on the UNC119 ability to dissociate G(t) subunits from each other and the membrane. We also found that UNC119 inhibited activation of G(o) by D2 dopamine receptor in cultured cells. Thus, UNC119 may play conserved inhibitory role in regulation of GPCR-G protein signaling in non-visual tissues.     

A new species of Isoperla (Insecta,Plecoptera) from the Karawanken,with considerations on the Southern Limestone Alps as centers of endemism

A new species of the genus Isoperla (Plecoptera, Perlodidae), belonging to the oxylepis species-group is described, and the male mating call is characterized. Its range falls within a small region of the Southern Limestone Alps which is well known to be one endemism-centre of aquatic insects.     

Electroretinographic study of the magnetic compass in European robins

Migratory birds are known to be sensitive to external magnetic field (MF). Much indirect evidence suggests that the avian magnetic compass is localized in the retina. Previously, we showed that changes in the MF direction could modulate retinal responses in pigeons. In the present study, we performed similar experiments using the traditional model animal to study the magnetic compass, European robins. The photoresponses of isolated retina were recorded using ex vivo electroretinography (ERG). Blue- and red-light stimuli were applied under an MF with the natural intensity and two MF directions, when the angle between the plane of the retina and the field lines was 0° and 90°, respectively. The results were separately analysed for four quadrants of the retina. A comparison of the amplitudes of the a- and b-waves of the ERG responses to blue stimuli under the two MF directions revealed a small but significant difference in a- but not b-waves, and in only one (nasal) quadrant of the retina. The amplitudes of both the a- and b-waves of the ERG responses to red stimuli did not show significant effects of the MF direction. Thus, changes in the external MF modulate the European robin retinal responses to blue flashes, but not to red flashes. This result is in a good agreement with behavioural data showing the successful orientation of birds in an MF under blue, but not under red illumination.     

Kinetic model of phosphofructokinase-1 from Escherichia coli

This paper presents a kinetic model of phosphofructokinase-1 from Escherichia coli. A complete catalytic cycle has been reconstructed based on available information on the oligomeric structure of the enzyme and kinetic mechanism of its monomer. Applying the generalization of the Monod-Wyman-Changeux approach proposed by Popova and Sel'kov(35-37) to the reconstructed catalytic cycle rate equation has been derived. Dependence of the reaction rate on pH, magnesium, and effectors has been taken into account. Kinetic parameters have been estimated via fitting the rate equation against experimentally measured dependencies of initial rate on substrates, products, effectors, and pH available from the literature. The model of phosphofructokinase-1 predicts (1) cooperativity of binding both fructose-6-phosphate and ATPMg(2-), (2) significant inhibition of the enzyme resulting from an increase in total concentration of ATP under the condition of fixed concentration of Mg(2+) ions, and (3) dual effect of ADP consisting of allosteric activation and product inhibition of the enzyme. Moreover, the model developed can be used in the kinetic modeling of biochemical pathways containing phosphofructokinase-1.     

In-stent restenosis after revascularization of myocardium with drug-eluting stents is accompanied by elevated level of blood plasma eosinophil cationic protein

The aim of this study was to assess the involvement of eosinophil cationic protein, a marker of eosinophil activation, in the development of in-stent restenosis after drug-eluting stent implantation. Follow-up angiography at 6 to 12?months was performed in 32 patients who were treated with percutaneous coronary intervention and implantation of sirolimus-eluting stents. Blood plasma levels of eosinophil cationic protein (ECP) and total immunoglobulin E (IgE) were measured by enzyme-linked immunosorbent assay and the level of C-reactive protein (hs-CRP) by high-sensitivity nephelometry. According to angiography data, in-stent restenosis occurred in 13 patients, while 19 patients did not develop it. There were no differences between the hs-CRP and IgE levels in patients with or without restenosis. In contrast, ECP level was higher in patients with restenosis compared with that in patients without restenosis [17.7?ng/mL (11.2-24.0) vs. 9.0?ng/mL (6.4-12.9), p?= 0.017]. The incidence of in-stent restenoses was 63% in patients with ECP level higher than or equal to 11?ng/mL, and 19% in patients with an ECP level lower than 11?ng/mL (p?= 0.019). These findings suggest that elevated eosinophil activation may play an important role in the pathogenesis of in-stent restenosis after implantation of drug-eluting stents.