Ultrafast conformational dynamics in cyclic azobenzene peptides of increased flexibility

Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the cis to trans and the trans to cis photoreactions. For the cis to trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the trans to cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps-1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10 ns.     

Three mechanisms of Red Queen dynamics

Models describing systems of coevolving populations often have asymptotically non-equilibrium dynamics (Red Queen dynamics (RQD)). We claim that if evolution is much slower than ecological changes, RQD arises due to either fast ecological processes, slow genetical processes, or to their interaction. The three corresponding generic types of RQD can be studied using singular perturbation theory and have very different properties and biological implications. We present simple examples of ecological, genetical, and ecogenetical RQD and describe how they may be recognized in natural populations. In particular, ecogenetical RQD often involve alternations of long epochs with radically different dynamics.     

Red queen dynamics of protein translation

We explore adaptive theories for the diversity of translational binding based on the genetic code viewed as a primitive mechanism of resistance. Modifying the set of codons bound by tRNA anticodon molecules or changing the specificity of binding, reduces the replication rate of translational parasites such as viruses. Increased translational efficiency of the parasite requires a high degree of specificity of host tRNAs for the parasite codons. This suggests that the genetic code might serve as the first line of defense against infection. We construct a red queen theory for translational diversity: a theory in which host-translational strategies- as defined by the degree of redundancy (a single anticodon binding many codons for a single amino acid) or degeneracy (many anticodons binding many codons for a single amino acid)-are constantly shifting through time to evade parasitism but where neither parasite nor host gain a systematic advantage.     

Seasonal dynamics of phytoplankton in the Gulf of Aqaba,Red Sea

Seawater samples were collected biweekly from the northern Gulf of Aqaba, Red Sea, for Phytoplankton analysis during the period May 1998 to October 1999. Microscopic counts and HPLC methods were employed. Procaryotic and eucaryotic ultraplankton dominated throughout most of the year, with larger nano- and microplankton making up only 5% of the photosynthetic biomass. Moderate seasonal variations in the 0–125 m integrated Chl a contrasted with a pronounced seasonal succession of the major taxonomic groups, reflecting the changes in the density stratification of the water column: Prochlorococcus dominated during the stratified summer period and were almost absent in winter. Chlorophyceae and Cryptophyceae were dominant during winter mixing but scarce or absent during summer. Diatoms and Synechococcus showed sharp and moderate biomass peaks in late winter and spring respectively, but remained at only low Chl a levels for the rest of the year. Chrysophyceae, Prymnesiophyceae and the scarce Dinophyceae showed no clear seasonal distribution pattern. The implications of alternating procaryotic and eucaryote dominated algal communities for the Red Sea pelagic food web are discussed. Electronic Supplementary Material Electronic supplementary material is available for this article at and accessible for authorised users.     

Membrane localization and dynamics of Nile Red: Effect of cholesterol

The organization and dynamics of the hydrophobic fluorescent probe Nile Red incorporated in DOPC vesicles containing varying amounts of cholesterol has been monitored utilizing fluorescence-based approaches which include the red edge excitation shift (REES) approach and the parallax method for depth determination. Our results show that the fluorescence emission maximum, intensity, polarization, and lifetime of Nile Red vary with the cholesterol content of the membrane. Interestingly, Nile Red exhibits significant REES independent of the presence of cholesterol. This indicates that Nile Red is localized in a motionally restricted environment in the membrane. This is supported by analysis of membrane penetration depth of Nile Red using the parallax method which points out to a membrane interfacial localization of Nile Red. These results could be useful in analyzing membrane organization and heterogeneity in natural membranes using Nile Red.     

Membrane localization and dynamics of Nile Red: effect of cholesterol

The organization and dynamics of the hydrophobic fluorescent probe Nile Red incorporated in DOPC vesicles containing varying amounts of cholesterol has been monitored utilizing fluorescence-based approaches which include the red edge excitation shift (REES) approach and the parallax method for depth determination. Our results show that the fluorescence emission maximum, intensity, polarization, and lifetime of Nile Red vary with the cholesterol content of the membrane. Interestingly, Nile Red exhibits significant REES independent of the presence of cholesterol. This indicates that Nile Red is localized in a motionally restricted environment in the membrane. This is supported by analysis of membrane penetration depth of Nile Red using the parallax method which points out to a membrane interfacial localization of Nile Red. These results could be useful in analyzing membrane organization and heterogeneity in natural membranes using Nile Red.     

Sequential regulation of the small GTPase Rap1 in human platelets

Rap1, a small GTPase of the Ras family, is ubiquitously expressed and particularly abundant in platelets. Previously we have shown that Rap1 is rapidly activated after stimulation of human platelets with alpha-thrombin. For this activation, a phospholipase C-mediated increase in intracellular calcium is necessary and sufficient. Here we show that thrombin induces a second phase of Rap1 activation, which is mediated by protein kinase C (PKC). Indeed, the PKC activator phorbol 12-myristate 13-acetate induced Rap1 activation, whereas the PKC-inhibitor bisindolylmaleimide inhibited the second, but not the first, phase of Rap1 activation. Activation of the integrin alpha(IIb)beta(3), a downstream target of PKC, with monoclonal antibody LIBS-6 also induced Rap1 activation. However, studies with alpha(IIb)beta(3)-deficient platelets from patients with Glanzmann's thrombasthenia type 1 show that alpha(IIb)beta(3) is not essential for Rap1 activation. Interestingly, induction of platelet aggregation by thrombin resulted in the inhibition of Rap1 activation. This downregulation correlated with the translocation of Rap1 to the Triton X-100-insoluble, cytoskeletal fraction. We conclude that in platelets, alpha-thrombin induces Rap1 activation first by a calcium-mediated pathway independently of PKC and then by a second activation phase mediated by PKC and, in part, integrin alpha(IIb)beta(3). Inactivation of Rap1 is mediated by an aggregation-dependent process that correlates with the translocation of Rap1 to the cytoskeletal fraction.     

Exploiting the potential of plant growth promoting bacteria in decolorization of dye Disperse Red 73 adsorbed on milled sugarcane bagasse under solid state fermentation

Bioremediation of textile dyes adsorbed on agricultural solid wastes under solid state fermentation (SSF) using rhizospheric plant growth promoting microorganisms pose an ecofriendly, economically feasible and promising treatment approach. The purpose of this study was to adsorb azo dye Disperse Red 73 (DR73) on sugarcane bagasse (SCB) and its further bioremediation using consortium-RARB under SSF. The particle size of SCB 0.002 mm showed maximum adsorption (65%) for DR73. Kinetics of adsorption of DR73 on milled SCB follows pseudo-second order kinetics. The individual cultures of Rhodobacter erythropholis MTCC 4688, Azotobacter vinelandii MTCC 1241, Rhizobium meliloti NCIM 2757 and Bacillus megaterium NCIM 2054 showed 44, 28, 50 and 61% decolorization of DR73 in 48 h respectively; while the consortium-RARB showed complete decolorization in 48 h. Optimum moisture content, temperature and pH for decolorization of DR73 was found to be 90%, 30 °C and 6 respectively. DR73 biodegradation analysis was carried out using HPTLC, FTIR and HPLC. Phytotoxicity and genotoxicity studies revealed detoxification of DR73. Tray bioreactor study for decolorization of adsorbed DR73 on SCB suggests its implementations at large scale. Use of plant growth promoting bacteria's consortium under SSF for bioremediation of adsorbed dyes gives a novel ecologically sustainable approach.     

Sequential 11 -hydroxylation and 1-dehydrogenation of 16 -hydroxycortexolone

Two-step microbial transformation of 16α-hydroxycortexolone to its 1-dehydro-11α-hydroxy derivative, without isolating an intermediate, was achieved with an overall yield of 72% of product at a steroid substrate concentration of 3 mg/ml. The process included formation of the cycloborate complex of the substrate, hydroxylation of the borate complex with a suspension of Aspergillus ochraceus mycelium in phosphate buffer, and dehydrogenation of the 11α-hydroxylated intermediate with acetone-dried Arthrobacter simplex cells. The desired product was then obtained by breaking the resultant borate complex through acidification.     

Electrophysiological evidence for push-pull interactions in the inner retina of turtle

The responses of the inner retinal neurons of turtle to light spots of sizes were studied in an attempt to reveal characteristics that may reflect possible interactions of the neural circuits underlying the center and surround responses. For the ON-OFF cells, the responses were also analyzed to observe whether interference or augmentation of these responses occur. The intracellular recordings revealed several such interactions, observed either in the form of altered spike activity or as changes in the transiency of the light responses. The ON-responding amacrine cell presented in this study became more sustained, while for the ON-OFF amacrine cells larger light spots tended to make the responses more transient and both the ON and OFF components became more pronounced. The spiking activity of the OFF-type ganglion cell shifted in relation to the light stimulus and the number of spikes observed upon presentation of larger spots increased. We suggest that the surround circuits activated by increasing light spots may substantially influence and reorganize not only the overall center-surround balance, but also the center response of the cells. Although it cannot be excluded that intrinsic membrane properties also influence these processes to some extent, it is more likely that lateral inhibition and disinhibitory mechanisms play the leading role in this process.     

Sequential unfolding of individual helices of bacterioopsin observed in molecular dynamics simulations of extraction from the purple membrane

Multiple molecular dynamics simulations of bacterioopsin pulling from its C-terminus show that its alpha-helices unfold individually. In the first metastable state observed in the simulations, helix G is unfolded at its C-terminal segment while the rest of helix G (residues 200-216) is folded and opposes resistance because of a salt-bridge network consisting of Asp-212 and Lys-216 on helix G and Arg-82 and Asp-85 on helix C. Helix G unfolds inside the bundle because the external force is applied to its C-terminal end in a direction perpendicular to the surface of the membrane. Inversely, helix F has to flip by 180 degrees to exit from the membrane because the applied force and the helical N-C axis point in opposite directions. At the highest peak of the force, which cannot be interpreted in single-molecule force spectroscopy experiments, helix F has a pronounced kink at Pro-186. Mutation of Pro-186 and/or the charged side chains mentioned above, which are involved in very favorable electrostatic interactions in the low-dielectric region of the membrane, are expected to reduce the highest peak of the force. Helices E and D unfold in a similar way to helices G and F, respectively. Hence, the force-distance profile and sequence of events during forced unfolding of bacterioopsin are influenced by the up-and-down topology of the seven-helix bundle. The sequential extraction of individual helices from the membrane suggests that the spontaneous (un)folding of bacterioopsin proceeds through metastable bundles of fewer than seven helices. The metastable states observed in the simulations provide atomic level evidence that corroborates the interpretation of very recent force spectroscopy experiments of bacteriorhodopsin refolding.     

Hill–Robertson interference maintained by Red Queen dynamics favours the evolution of sex

Although it is well established theoretically that selective interference among mutations (Hill–Robertson interference) favours meiotic recombination, genomewide mean rates of mutation and strengths of selection appear too low to support this as the mechanism favouring recombination in nature. A possible solution to this discrepancy between theory and observation is that selection is at least intermittently very strong due to the antagonistic coevolution between a host and its parasites. The Red Queen theory posits that such coevolution generates fitness epistasis among loci, which generates negative linkage disequilibrium among beneficial mutations, which in turn favours recombination. This theory has received only limited support. However, Red Queen dynamics without epistasis may provide the ecological conditions that maintain strong and frequent selective interference in finite populations that indirectly selects for recombination. This hypothesis is developed here through the simulation of Red Queen dynamics. This approach required the development of a method to calculate the exact frequencies of multilocus haplotypes after recombination. Simulations show that recombination is favoured by the moderately weak selection of many loci involved in the interaction between a host and its parasites, which results in substitution rates that are compatible with empirical estimates. The model also reproduces the previously reported rapid increase in the rate of outcrossing in Caenorhabditis elegans coevolving with a bacterial pathogen.     

Conformational dynamics of the F1-ATPase beta-subunit: a molecular dynamics study

According to the different nucleotide occupancies of the F(1)-ATPase beta-subunits and due to the asymmetry imposed through the central gamma-subunit, the beta-subunit adopts different conformations in the crystal structures. Recently, a spontaneous and nucleotide-independent closure of the open beta-subunit upon rotation of the gamma-subunit has been proposed. To address the question whether this closure is dictated by interactions to neighbored subunits or whether the open beta-subunit behaves like a prestressed "spring," we report multinanosecond molecular dynamics simulations of the isolated beta-subunit with different start conformations and different nucleotide occupancies. We have observed a fast, spontaneous closure motion of the open beta(E)-subunit, consistent with the available x-ray structures. The motions and kinetics are similar to those observed in simulations of the full (alpha beta)(3)gamma-complex, which support the view of a prestressed "spring," i.e., that forces internal to the beta(E)-subunit dominate possible interactions from adjacent alpha-subunits. Additionally, nucleotide removal is found to trigger conformational transitions of the closed beta(TP)-subunit; this provides evidence that the recently resolved half-closed beta-subunit conformation is an intermediate state before product release. The observed motions provide a plausible explanation why ADP and P(i) are required for the release of bound ATP and why gamma-depleted (alpha beta)(3) has a drastically reduced hydrolysis rate.     

On the intra-host dynamics of HIV-1 infections

An extension of a previously proposed theory for the pathogenesis of AIDS is presented and analyzed using a mathematical modelling approach. This theory is based on the observation that human immunodeficiency virus type 1 (HIV-1) predominantly infects and replicates in (CD4+)-T cells, and that the infection process within an infected individual is characterized by ongoing generation and selection of HIV variants with increasing reproductive capacity. This evolutionary process is considered to be the reason for the gradual loss of immunocompetence and the final destruction of the immune system observed in most patients. The extension presented here incorporates the effect of the permanently increasing susceptibility of (CD4+)-T cell clones, as a result of the evolutionary process. The presented model reproduces and possibly explains a wide variety of findings about the HIV infection process. Numerical results indicate that the effect of the initial dose is minimal, and restricted to the primary phase of infection. According to the model predictions the impact of the HIV evolutionary speed is crucial for the progression to disease. An important progression determinant is the initial infection rate, being a component of the viral reproductive capacity. An influential role in disease progression seems to be played by the initial (CD4+)-T cell count.