An in-silico study of alphaherpesviruses ICP0 genes: positive selection or strong mutational GC-pressure?

The purpose of our work was to analyze the case of the strong mutational GC-pressure influence on the ratio between nonsynonymous (DN) and synonymous (DS) distances (DN/DS ratio). We have used as the material the genes coding for ICP0 from five completely sequenced genomes of simplexviruses. DN/DS ratio, total GC-content (G + C), and GC-content in first, second, and third codon positions (1GC, 2GC, and 3GC, respectively) have been calculated separately for exon 2, nonconserved part of exon 3, and conserved part of exon 3 from ICP0 genes. Results showed that DN is more than DS only in the conserved part of exon 3 of ICP0 genes from cercopithecine herpesvirus 2 and cercopithecine herpesvirus 16. However, the cause of this result (DN/DS = 2.54) is the GC-pressure acting on the coding districts with 3GC = 99% rather than the biological process called positive selection. Only in these two viruses, because of the strong GC-pressure, 3GC has reached 99% in the conserved part of ICP0 exon 3, and so nucleotide substitutions that increase the GC-content practically cannot occur in third codon positions, where most substitutions are synonymous. In this case, GC-pressure has a substrate for nucleotide substitutions only in first and second codon positions, where most substitutions are nonsynonymous.     

Sialylation in protostomes: a perspective from <Emphasis Type="Italic">Drosophila</Emphasis> genetics and biochemistry

Numerous studies have revealed important functions for sialylation in both prokaryotes and higher animals. However, the genetic and biochemical potential for sialylation in Drosophila has only been confirmed recently. Recent studies suggest significant similarities between the sialylation pathways of vertebrates and insects and provide evidence for their common evolutionary origin. These new data support the hypothesis that sialylation in insects is a specialized and developmentally regulated process which likely plays a prominent role in the nervous system. Yet several key issues remain to be addressed in Drosophila, including the initiation of sialic acid de novo biosynthesis and understanding the structure and function of sialylated glycoconjugates. This review discusses our current knowledge of the Drosophila sialylation pathway, as compared to the pathway in bacteria and vertebrates. We arrive at the conclusion that Drosophila is emerging as a useful model organism that is poised to shed new light on the function of sialylation not only in protostomes, but also in a larger evolutionary context. K. Koles and E. Repnikova contributed equally to this work.     

His-75 in Proteorhodopsin, a Novel Component in Light-driven Proton Translocation by Primary Pumps

Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near the photoactive site of the protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements of light-induced charge movements reveal participation of His-75 in the proton translocation mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the structure of the photoactive site and resulted in significantly shifted visible absorption spectra. In contrast, His-75 substitution with a positively charged Arg did not shift the visible absorption spectrum of PR. The mutation to Arg also blocks the light-induced proton transfer from the Schiff base to its counterion Asp-97 during the photocycle and the acid-induced protonation of Asp-97 in the dark state of the protein. Isotope labeling of histidine revealed that His-75 undergoes deprotonation during the photocycle in the proton-pumping (high pH) form of PR, a reaction further supported by results from H75E. Finally, all His-75 mutations greatly affect charge movements within the PR and shift its pH dependence to acidic values. A model of the proteorhodopsin proton transport process is proposed as follows: (i) in the dark state His-75 is positively charged (protonated) over a wide pH range and interacts directly with the Schiff base counterion Asp-97; and (ii) photoisomerization-induced transfer of the Schiff base proton to the Asp-97 counterion disrupts its interaction with His-75 and triggers a histidine deprotonation.A variety of unicellular microorganisms contain primary proton pumps that convert solar energy into a transmembrane electrochemical proton gradient, which is subsequently used by membrane ATP synthases to generate chemical energy. Well known examples of such pumps are the haloarchaeal rhodopsins, photoactive, seven-helix membrane proteins, which include the well studied proton pump bacteriorhodopsin (BR)⁴ from Halobacterium salinarum and BR homologs in other haloarchaea. Recently, a much larger new family of light-driven proton pumps, the proteorhodopsins (PRs), was identified in marine proteobacteria throughout the oceans (1–3). Despite the diverse properties of PRs, including different visible absorption maxima and photocycle rates (4–6), they all share with BR several key conserved residues as well as an all-trans-retinylidene chromophore in their unphotolyzed state, which is covalently bound to transmembrane helix G via a protonated Schiff base linkage.Many of the molecular events that occur in PRs following light activation are similar to those of BR, including an initial ultrafast all-trans→13-cis-retinal isomerization, which triggers a sequence of protein conformational changes, including several intramolecular proton transfer reactions. The two key carboxylate groups involved in proton pumping in helix C of BR are conserved in PRs, and in the first found and most commonly studied PR, the Monterey Bay variant eBAC31A08, also known as green-absorbing proteorhodopsin (GPR), the helix C residues Asp-97 and Glu-108 undergo protonation changes during the photocycle similar to those of the homologous carboxylate residues in BR. Initial FTIR studies on GPR identified the role of Asp-97 as the Schiff base counterion and proton acceptor during Schiff base deprotonation and concomitant M formation and Glu-108 as the proton donor that reprotonates the Schiff base during N formation (7, 8). Studies of other variants indicate these roles of the two carboxylic acid residues are general in the proteorhodopsin family.⁵One major difference between BR and the PRs is the presence of a highly conserved histidine residue at position 75, near the middle of transmembrane helix B in the latter pigments. The His-75 homolog is not present in BR nor thus far found in other microbial rhodopsins (9). The proximity of His-75 to the protein active site and specifically to the Schiff base counterion Asp-97 inferred from the x-ray crystal structure of BR suggests its involvement in spectral tuning of the visible absorption (10) and potentially PR photochemical reactions. Because the pK_a of histidine in solution is close to neutral pH (11), its imidazole group often plays a major role in intramolecular proton transfers in enzymes, including NADPH oxidase (12), alcohol dehydrogenase (13), carbonic anhydrase II (14), and serine proteases (15).In this study we have used a combination of time-resolved FTIR difference spectroscopy, visible absorption spectroscopy, isotope labeling, kinetic charge displacement measurements, and site-directed mutagenesis to study the role of His-75 in GPR. We report evidence that protonated His-75 interacts directly with Asp-97 in the unphotolyzed protein and during the photocycle undergoes a deprotonation in response to the protonation of Asp-97.     

Variation in host specificity between species of avian hemosporidian parasites: evidence from parasite morphology and cytochrome B gene sequences

A parasite's shift to a new host may have serious evolutionary consequences, since host switching usually is associated with a change in virulence and may lead to the evolution of emerging diseases. This phenomenon remains insufficiently studied in wildlife. Here, we combine microscopic examination of blood films and PCR-based methods to investigate the natural host specificity of Haemoproteus and Plasmodium spp. in birds of 4 families of the Passeriformes within a small geographic area. The material was collected on the Curonian Spit in the Baltic Sea between May and July in 2003-2004. A nested-PCR protocol was used for amplifying and sequencing a fragment of 480 nucleotides of the cytochrome b gene of the mtDNA of these parasites. Blood samples from 282 birds, which were positive both by microscopic examination of blood films and mtDNA amplification, were used in this study. We found that Haemoproteus majoris (lineages hPARUS1, hCCF5, hWW2, and hPHSIB1), Haemoproteus sp. (hWW1), Plasmodium (Haemamoeba) sp. (pSGS1), and Plasmodium (Haemamoeba) sp. (pGRW11) are capable of infecting birds belonging to different families of passeriform birds. Some species of Haemoproteus are less specific than have been traditionally believed. Haemoproteus majoris appears to have a genetic predisposition to have a broad host range. The level of host specificity varies markedly among different species of hemosporidian parasites of birds. The natural host range is thus not a reliable taxonomic character in the systematics of these parasites in the form in which it is still accepted in some recent taxonomic studies.     

Backbone and ILV side chain methyl group assignments of the integral human membrane protein VDAC-1

The voltage dependent anion channel (VDAC) forms a channel for metabolites and nutrients in the outer membrane of mitochondria, and it is also involved in apoptotic pathways. Here, we report sequence-specific NMR assignments for the isoform 1 of human VDAC reconstituted in lauryldimethylamine oxide (LDAO) detergent micelles. The assignments were deposited in the BMRB data base with accession number 16381.     

Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells

Localization-based superresolution optical imaging is rapidly gaining popularity, yet limited availability of genetically encoded photoactivatable fluorescent probes with distinct emission spectra impedes simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric photoactivatable far-red fluorescent protein, which facilitates simultaneous imaging of three photoactivatable proteins in mammalian cells using fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning only ∼100 nm of the visible spectrum. Raft-, non-raft-, and cytoskeleton-associated proteins were simultaneously imaged in both live and fixed fibroblasts coexpressing Dendra2-hemagglutinin, PAmKate-transferrin receptor, and PAmCherry1-β-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures.     

Mechanisms of atrial-selective block of Na⁺ channels by ranolazine: II. Insights from a mathematical model

Block of Na(+) channel conductance by ranolazine displays marked atrial selectivity that is an order of magnitude higher that of other class I antiarrhythmic drugs. Here, we present a Markovian model of the Na(+) channel gating, which includes activation-inactivation coupling, aimed at elucidating the mechanisms underlying this potent atrial selectivity of ranolazine. The model incorporates experimentally observed differences between atrial and ventricular Na(+) channel gating, including a more negative position of the steady-state inactivation curve in atrial versus ventricular cells. The model assumes that ranolazine requires a hydrophilic access pathway to the channel binding site, which is modulated by both activation and inactivation gates of the channel. Kinetic rate constants were obtained using guarded receptor analysis of the use-dependent block of the fast Na(+) current (I(Na)). The model successfully reproduces all experimentally observed phenomena, including the shift of channel availability, the sensitivity of block to holding or diastolic potential, and the preferential block of slow versus fast I(Na.) Using atrial and ventricular action potential-shaped voltage pulses, the model confirms significantly greater use-dependent block of peak I(Na) in atrial versus ventricular cells. The model highlights the importance of action potential prolongation and of a steeper voltage dependence of the time constant of unbinding of ranolazine from the atrial Na(+) channel in the development of use-dependent I(Na) block. Our model predictions indicate that differences in channel gating properties as well as action potential morphology between atrial and ventricular cells contribute equally to the atrial selectivity of ranolazine. The model indicates that the steep voltage dependence of ranolazine interaction with the Na(+) channel at negative potentials underlies the mechanism of the predominant block of I(Na) in atrial cells by ranolazine.     

The pathway and spatial scale for MscS inactivation

The mechanosensitive channel of small conductance (MscS) is a bacterial tension-driven osmolyte release valve with homologues in many walled eukaryotic organisms. When stimulated by steps of tension in excised patches, Escherichia coli MscS exhibits transient opening followed by reversible adaptation and then complete inactivation. Here, we study properties of the inactivation transition, which renders MscS nonconductive and tension insensitive. Using special pressure protocols we demonstrate that adaptation and inactivation are sequential processes with opposite tension dependencies. In contrast to many eukaryotic channels, which inactivate from the open state, MscS inactivates primarily from the closed state because full openings by preconditioning pulses do not influence the degree of inactivation, and saturating tensions keeping channels open prevent inactivation. The easily opened A98S mutant lacks inactivation completely, whereas the L111S mutant with a right-shifted activation curve inactivates silently before reaching the threshold for opening. This suggests that opening and inactivation are two independent tension-driven pathways, both starting from the closed state. Analysis of tension dependencies for inactivation and recovery rates estimated the in-plane expansion (ΔA) associated with inactivation as 8.5 nm(2), which is about half of the area change for opening. Given that the interhelical contact between the outer TM1-TM2 pairs and the core TM3s is the force-transmitting path from the periphery to the gate, the determined ΔA now can be used as a constraining parameter for the models of the inactivated state in which the lipid-facing TM1-TM2 pairs are displaced and uncoupled from the gate.