The dynamics of a quantitative trait in an age-structured population living in a variable environment

Time series of rapid phenotypic change have been documented in age-structured populations living in the wild. Researchers are often interested in identifying the processes responsible for such change. We derive an equation to exactly decompose change in the mean value of a phenotypic trait into contributions from fluctuations in the demographic structure and age-specific viability selection, fertility selection, phenotypic plasticity, and differences between offspring and parental trait values. We treat fitness as a sum of its components rather than as a scalar and explicitly consider age structure by focusing on short time steps, which are appropriate for describing phenotypic change in species with overlapping generations. We apply the method to examine stasis in birth weight in a well-characterized population of red deer. Stasis is achieved because positive viability selection for an increase in birth weight is countered by parents producing offspring that are, on average, smaller than they were at birth. This is one of many ways in which equilibria in the mean value of a phenotypic trait can be maintained. The age-structured Price equation we derive has the potential to provide considerable insight into the processes generating now frequently reported cases of rapid phenotypic change.     

Dynamic structure of disulfide-removed linear analogs of tachyplesin-I in the lipid bilayer from solid-state NMR

Tachyplesin-I (TP-I) is a 17-residue beta-hairpin antimicrobial peptide containing two disulfide bonds. Linear analogs of TP-I where the four Cys residues were replaced by aromatic and aliphatic residues, TPX4, were found to have varying degrees of activities, with the aromatic analogs similarly potent as TP-I. Understanding the different activities of the linear analogs should give insight into the mechanism of action of TP-I. To this end, we have investigated the dynamic structures of the active TPF4 and the inactive TPA4 in bacteria-mimetic anionic POPE/POPG bilayers and compared them with the wild-type TP-I using solid-state NMR spectroscopy. 13C isotropic chemical shifts and backbone (phi, psi) torsion angles indicate that both TPF4 and TPA4 adopt beta-strand conformations without a beta-turn at key residues. 1H spin diffusion from lipid chains to the peptide indicates that the inactive TPA4 binds to the membrane-water interface, similar to the active TP-I. Thus, neither the conformation nor the depth of insertion of the three peptides correlates with their antimicrobial activities. In contrast, the mobility of the three peptides correlates well with their activities: the active TP-I and TPF4 are both highly mobile in the liquid-crystalline phase of the membrane while the inactive TPA4 is completely immobilized. The different mobilities are manifested in the temperature-dependent 13C and 15N spectra, 13C-1H and 15N-1H dipolar couplings and 1H rotating-frame spin-lattice relaxation times. The dynamics of TP-I and TPF4 are both segmental and global. Combined, these data suggest that TP-I and TPF4 disrupt the membrane by large-amplitude motion in the plane of the membrane. The loss of this motion in TPA4 due to aggregation significantly weakens its activity because a higher peptide concentration is required to disturb lipid packing. Thus molecular motion, rather than structure, appears to be the key determinant for the membrane-disruptive activities of tachyplesins.     

Plk1 activation by Ste20-like kinase (Slk) phosphorylation and polo-box phosphopeptide binding assayed with the substrate translationally controlled tumor protein (TCTP)

The mitotic protein kinase Plk1 catalyzes events associated with centrosome maturation, kinetocore function, spindle formation, and cytokinesis and is a target for anticancer drug design. It is composed of a N-terminal kinase domain and a C-terminal polo-box domain (PBD). The PBD domain serves to localize the kinase on cognate phosphorylated substrates, and this binding relieves the inhibition of the kinase by the PBD. Similar to many protein kinases, Plk1 is activated by phosphorylation on a threonine residue, Thr210, in the activation segment. In this work, we describe expression in Escherichia coli cells and purification of full-length Plk1 in quantities suitable for structural studies and use this material for quantitative characterization of the activation events with the substrate translationally controlled tumour protein (TCTP). The presence of the PBD-binding phosphopeptide enhances phosphorylation by the activating Ste20-like kinase (Slk). Native Plk1 exhibits a basal catalytic efficiency k cat/ K(M) of 9.9 x 10 (-5) s (-1) microM (-1). Association with a polo-box-binding phosphopeptide increased the catalytic efficiency by 11x largely through an increase in k(cat) with no change in K(M). Phosphorylation by Slk increases catalytic efficiency by 202x with a 2.3-fold reduction in K(M) and 88-fold increase in k(cat). Phosphorylation and the presence of the PBD-binding phosphopeptide result in an increase in catalytic efficiency of 1515x with a 2.3-fold decrease in K(M) and a 705-fold increase in k(cat) over the unmodified Plk1. Knowledge of kinase regulatory mechanisms and the structures of the Plk1 individual domains has allowed for a model to be proposed for these activatory events.     

Design and investigation of a Ru(II) N-heterocyclic complex which undergoes proton coupled electron transfer

A novel tridentating ligand containing a single ionizable proton was designed for studying proton coupled electron transfer. The ligand was synthesized by derivatizing 2,2′-bipyridine at the 6-position with benzimidazole (bpy-bzimH), and it was used to prepare the compound [Ru(bpy-bzimH)₂](PF₆)₂. Cyclic voltammetry was used to characterize the redox behavior in an aprotic (0.1 M TBAH-CH₂Cl₂) and protic (1:1 acetonitrile-water buffered solutions) solvent conditions where the latter was employed to characterize the pH-dependence of the Ru(III/II) couple. The redox potential as a function of pH was plotted and reveals a one-proton/one-electron transfer in two separate pH regions (1.39-2.58, and 5.92-7.97), while a two-proton/one-electron process was exhibited between 2.58 and 5.92.     

Homodimeric theta-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides.

Rhesus theta-defensin 1 (RTD-1) is a unique tridisulfide, cyclic antimicrobial peptide formed by the ligation of two 9-residue sequences derived from heterodimeric splicing of similar 76-amino acid, alpha-defensin-related precursors, termed RTD1a and RTD1b (Tang, Y. Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C. J., Ouellette, A. J., and Selsted, M. E. (1999) Science 286, 498-502). The structures of RTD-2 and RTD-3 were predicted to exist if homodimeric splicing of the RTD1a and RTD1b occurs in vivo. Western blotting disclosed the presence of putative theta-defensins, distinct from RTD-1, in leukocyte extracts. Two new theta-defensins, RTD-2 and RTD-3, were purified by reverse-phase high performance liquid chromatography and characterized by amino acid analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, and comparison to the synthetic standards. RTD-2 and RTD-3 are the predicted homodimeric splicing products of RTD1b and RTD1a, respectively. The cellular abundances of RTD-1, -2, and -3 were 29:1:2, indicating that there is a preference for the heterodimeric ligation that generates RTD-1. RTD-1, -2, and -3 had similar antimicrobial activities against Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans, whereas the activity of RTD-2 against Escherichia coli was 2-3-fold less than those of RTD-1 and RTD-3. Equal amounts of each theta-defensin bound to E. coli cells, indicating that the differences in antibacterial activities are the result of post-binding processes.     

Functional differences between TRPC4 splice variants.

Functional characterizations of heterologously expressed TRPC4 have revealed diverse regulatory mechanisms and permeation properties. We aimed to clarify whether these differences result from different species and splice variants used for heterologous expression. Like the murine beta splice variant, rat and human TRPC4beta both formed receptor-regulated cation channels when expressed in HEK293 cells. In contrast, human TRPC4alpha was poorly activated by stimulation of an H(1) histamine receptor. This was not due to reduced expression or plasma membrane targeting, because fluorescent TRPC4alpha fusion proteins were correctly inserted in the plasma membrane. Furthermore, currents through both human TRPC4alpha and TRPC4beta had similar current-voltage relationships and single channel conductances. To analyze the assembly of transient receptor potential channel subunits in functional pore complexes in living cells, a fluorescence resonance energy transfer (FRET) approach was used. TRPC4alpha and TRPC4beta homomultimers exhibited robust FRET signals. Furthermore, coexpressed TRPC4alpha and TRPC4beta subunits formed heteromultimers exhibiting comparable FRET signals. To promote variable heteromultimer assemblies, TRPC4alpha/TRPC4beta were coexpressed at different molar ratios. TRPC4beta was inhibited in the presence of TRPC4alpha with a cooperativity higher than 2, indicating a dominant negative effect of TRPC4alpha subunits in heteromultimeric TRPC4 channel complexes. Finally, C-terminal truncation of human TRPC4alpha fully restored the channel activity. Thus, TRPC4beta subunits form a receptor-dependently regulated homomultimeric channel across various species, whereas TRPC4alpha contains a C-terminal autoinhibitory domain that may require additional regulatory mechanisms.     

Structure and function of threonine synthase from yeast.

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 A resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open alpha/beta architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the beta-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.     

Stem water transport and freeze-thaw xylem embolism in conifers and angiosperms in a Tasmanian treeline heath

The effect of freezing on stem xylem hydraulic conductivity and leaf chlorophyll a fluorescence was measured in 12 tree and shrub species from a treeline heath in Tasmania, Australia. Reduction in stem hydraulic conductivity after a single freeze-thaw cycle was minimal in conifers and the vessel-less angiosperm species Tasmannia lanceolata (Winteraceae), whereas mean loss of conductivity in vessel-forming angiosperms fell in the range 17-83%. A positive linear relationship was observed between percentage loss of hydraulic conductivity by freeze-thaw and the average conduit diameter across all 12 species. This supports the hypothesis that large-diameter vascular conduits have a greater likelihood of freeze-thaw cavitation because larger bubbles are produced, which are more likely to expand under tension. Leaf frost tolerances, as measured by a 50% loss of maximum PSII quantum yield, varied from -6 to -13°C, indicating that these species were more frost-sensitive than plants from northern hemisphere temperate forest and treeline communities. There was no evidence of a relationship between frost tolerance of leaves and the resilience of stem water transport to freezing, suggesting that low temperature survival and the resistance of stem water transport to freezing are independently evolving traits. The results of this study bear on the ecological importance of stem freezing in the southern hemisphere treeline zones.