Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)(10) using solid-state NMR methods

Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), ¹³C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of ¹³C=O isotope-labeled (Ala-Gly-Gly)₁₀. The torsion angles were determined to be (, ) = (–90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 3₁ helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 3₁ helix of (Ala-Gly-Gly)₁₀ does not change by formic acid treatment although (Ala-Gly)₁₅ easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 3₁ helix conformation of (Ala-Gly-Gly)₁₀ is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)₁₀ were also determined as (, ) = (–90°, 150° ) and this peptide is also considered to take 3₁ helix conformation.     

Thermodynamic and structural characterization of Asn and Ala residues in the disallowed II' region of the Ramachandran plot

Residue Asn47 at position L1 of a type II' beta-turn of the alpha-spectrin SH3 domain is located in a disallowed region of the Ramachandran plot (phi = 56 +/- 12, psi = -118 +/- 17). Therefore, it is expected that replacement of Asn47 by Gly should result in a considerable stabilization of the protein. Thermodynamic analysis of the N47G and N47A mutants shows that the change in free energy is small (approximately 0.7 kcal/mol; approximately 3 kJ/mol) and comparable to that found when mutating a Gly to Ala in a alpha-helix or beta-sheet. X-ray structural analysis of these mutants shows that the conformation of the beta-turn does not change upon mutation and, therefore, that there is no relaxation of the structure, nor is there any gain or loss of interactions that could explain the small energy change. Our results indicate that the energetic definition of II' region of the Ramachandran plot (phi = 60 +/- 30, psi = -115 +/- 15) should be revised for at least Ala and Asn in structure validation and protein design.     

Applications of likelihood asymptotics for nonlinear regression in herbicide bioassays

Dose-response models are intensively used in herbicide bioassays. Despite recent advancements in the development of new herbicides, statistical analyses are commonly based on asymptotic approximations that are sometimes poor. This paper presents the use of recent results in higher order asymptotics for likelihood-based inference in nonlinear regression. The methods presented provide accurate approximation for the distribution of test statistics and for prediction limits. Analyses of the fit and measures of detection limits of the bioassays are considered, and the potential of the methods is illustrated by examples with real data.     

Short-term and long-term effects of mowing on the vegetation of two calcareous fens

Abstract. Short-term field experiments are often used to predict and evaluate long-term management effects. Based on a mowing experiment in two calcareous fens near Lake Neuchâtel, Switzerland, we investigated whether short-term treatment effects (i.e. during the first four years) were confirmed by long-term results (13 - 14 yr). Plots were mown in summer or in winter or left unmown. The main long-term trends in overall species composition (based on percentage cover estimates) were already observable in the first four years: mown and unmown plots diverged, whereas summer-cut and winter-cut plots remained similar. At the individual species level, however, short-term and long-term treatment effects differed considerably: many species whose abundance seemed affected by treatments during the first four years showed no response in the long term, and vice versa. These discrepancies were similar when based on cover estimates or on counts of shoots. Species responses did actually depend on the time scale considered. Short-term and long-term treatment effects on species richness were similar (i.e. a decrease in unmown plots), although only long-term effects were significant. Treatment effects on the above-ground biomass varied considerably, and short-term trends (lower biomass in unmown plots) differed from long-term trends (higher biomass in unmown plots). Our sites showed little overall change in species composition during the period investigated, and treatment effects were low compared with other similar experiments. If study sites are less stable or treatment effects more drastic, a short-term evaluation is expected to be even less reliable. Knowledge on species dynamics at a site may help to choose the adequate spatial and temporal scale of investigation, and thus increase the efficiency of management experiments.     

A test of the mechanisms behind avian generalized individuals–area relationships

Aim Questions related to abundances of organisms are central to ecological research. A priori, a scale independent estimation of abundances would be expected. However, we find estimates of numbers of bird individuals from all over the world to increase less than proportionately with increasing plot size. At the whole assemblage level, the pattern holds across biogeographical regions and habitats. The slope of the interspecific and, for the majority of species, the intraspecific individuals–area relationship is also significantly shallower than 1. The question arises as to which mechanisms cause these patterns. Location Global. Methods At the assemblage, interspecific and intraspecific levels, we tested three mechanisms that could be responsible for these patterns by comparing the slope of the individuals–plot area relationship for subsets of a database compiled from the literature. Spatial autocorrelation was controlled for. Results There was no evidence for an influence of plot area choice in order to sample a constant number of individuals. Evidence for higher survey efficiency was available only with increasing number of visits at the intraspecific level. Evidence for influences of habitat heterogeneity was present at the assemblage, interspecific and intraspecific levels. This mechanism can work only if small plots are delimited non‐randomly in homogeneous habitat. Main conclusions Avian population size estimates without indication of the area over which they were obtained are of substantially less value than those coupled with that information. Ecologists planning to compare avian abundances between plots varying in some other factor of interest should minimize variations in their areas and/or account for them in data analyses. Population viability analyses, regional and global population size estimates, site prioritization and the scaling of ecosystem and species energy utilization need to address the plot area effect on assemblage and individual species abundances.     

Comparison of dwarf bamboos (Indocalamus sp.) leaf parameters to determine relationship between spatial density of plants and total leaf area per plant

The relationship between spatial density and size of plants is an important topic in plant ecology. The self‐thinning rule suggests a ?3/2 power between average biomass and density or a ?1/2 power between stand yield and density. However, the self‐thinning rule based on total leaf area per plant and density of plants has been neglected presumably because of the lack of a method that can accurately estimate the total leaf area per plant. We aimed to find the relationship between spatial density of plants and total leaf area per plant. We also attempted to provide a novel model for accurately describing the leaf shape of bamboos. We proposed a simplified Gielis equation with only two parameters to describe the leaf shape of bamboos one model parameter represented the overall ratio of leaf width to leaf length. Using this method, we compared some leaf parameters (leaf shape, number of leaves per plant, ratio of total leaf weight to aboveground weight per plant, and total leaf area per plant) of four bamboo species of genus Indocalamus Nakai (I. pedalis (Keng) P.C. Keng, I. pumilus Q.H. Dai and C.F. Keng, I. barbatus McClure, and I. victorialis P.C. Keng). We also explored the possible correlation between spatial density and total leaf area per plant using log‐linear regression. We found that the simplified Gielis equation fit the leaf shape of four bamboo species very well. Although all these four species belonged to the same genus, there were still significant differences in leaf shape. Significant differences also existed in leaf area per plant, ratio of leaf weight to aboveground weight per plant, and leaf length. In addition, we found that the total leaf area per plant decreased with increased spatial density. Therefore, we directly demonstrated the self‐thinning rule to improve light interception.     

Time-resolved FRET reveals the structural mechanism of SERCA–PLB regulation

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca²⁺ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca²⁺]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca²⁺ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca²⁺ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex.     

Is glycine a surrogate for a d‐amino acid in the collagen triple helix?

Collagen is the most abundant protein in animals. Every third residue in a collagen strand is a glycine with phi, psi = -70 degrees, 175 degrees. A recent computational study suggested that replacing these glycine residues with D-alanine or D-serine would stabilize the collagen triple helix. This hypothesis is of substantial importance, as the glycine residues in collagen constitute nearly 10% of the amino acid residues in humans. To test this hypothesis, we synthesized a series of collagen mimic peptides that contain one or more D-alanine or D-serine residues replacing the canonical glycine residues. Circular dichroism spectroscopy and thermal denaturation experiments indicated clearly that the substitution of glycine with D-alanine or D-serine greatly disfavors the formation of a triple helix. Host-guest studies also revealed that replacing a single glycine residue with D-alanine is more destabilizing than is its replacement with L-alanine, a substitution that results from a common mutation in patients with collagen-related diseases. These data indicate that the glycine residues in collagen are not a surrogate for a D-amino acid and support the notion that the main-chain torsion angles of a glycine residue in the native structure (especially, phi > 0 degrees ) are critical determinants for its beneficial substitution with a D-amino acid in a protein.     

Steady-state analysis of metabolic pathways: comparing the double modulation method and the lin-log approach

The increasing interest in studying enzyme kinetics under in vivo conditions requires practical methods to estimate control parameters from experimental data. In contrast to currently established approaches of dynamic modelling, this paper addresses the steady-state analysis of metabolic pathways. Within the framework of metabolic control analysis (MCA), elasticity coefficients are used to describe the control properties of a local enzyme reaction. The double modulation method is one of the first experimental approaches to estimate elasticity coefficients from measurements of steady-state flux rates and metabolite concentrations. We propose a generalized form of the double modulation method and compare it to the recently developed linear-logarithmic approach.