Structural features of the combining site region of Erythrina corallodendron lectin: role of tryptophan 135.

The role of Trp 135 and Tyr 108 in the combining site of Erythrina corallodendron lectin (ECorL) was investigated by physicochemical characterization of mutants obtained by site-directed mutagenesis, hemagglutination-inhibition studies, and molecular modeling, including dynamics simulations. The findings demonstrate that Trp 135 in ECorL: (1) is required for the tight binding of Ca2+ and Mn2+ to the lectin because mutation of this residue into alanine results in loss of these ions upon dialysis and concomitant reversible inactivation of the mutant; (2) contributes to the high affinity of methyl alpha-N-dansylgalactosaminide (MealphaGalNDns) to the lectin; and (3) is solely responsible for the fluorescence energy transfer between the aromatic residues of the lectin and the dansyl group in the ECorL-MealphaGalNDns complex. Docking of MealphaGalNDns into the combining site of the lectin reveals that the dansyl moiety is parallel with the indole of Trp 135, as required for efficient fluorescence energy transfer, in one of the two possible conformations that this ligand assumes in the bound state. In the W135A mutant, which still binds MealphaGalNDns strongly, the dansyl group may partially insert itself into the place formerly occupied by Trp 135, a process that from dynamics simulations does not appear to be energetically favored unless the loop containing this residue assumes an open conformation. However, a small fraction of the W135A molecules must be able to bind MealphaGalNDns in order to explain the relatively high affinity, as compared to galactose, still remaining for this ligand. A model for the molecular events leading to inactivation of the W135A mutant upon demetallization is also presented in which the cis-trans isomerization of the Ala 88-Asp 89 peptide bond, observed in high-temperature dynamics simulations, appears not to be a required step.     

Body size influences differently the detectabilities of colour morphs of cryptic prey

Body size and coloration may contribute to variation in performance and fitness among individuals; for example, by influencing vulnerability to predators. Yet, the combined effect of size and colour pattern on susceptibility to visual predators has received little attention, particularly in camouflaged prey. In the colour polymorphic pygmy grasshopper Tetrix subulata (Linnaeus, 1758), females are larger than males, although there is a size overlap between sexes. In the present study, we investigated how body size and colour morph influenced detection of these grasshoppers, and whether differences in protective value among morphs change with size. We conducted a computer‐based experiment and compared how human ‘predators’ detected images of large, intermediate or small grasshoppers belonging to black, grey or striped colour morphs when embedded in photographs of natural grasshopper habitats. We found that time to detection increased with decreasing size, that differences in time to detection of the black, grey and striped morphs depended differently on body size, and that no single morph provided superior or inferior protection in all three size classes. By comparing morph frequencies in samples of male and female grasshoppers from natural populations, we also examined whether the joint effects of size and colour morph on detection could explain evolutionary dynamics in the wild. Morph frequency differences between sexes were largely in accordance with expectations from the results of the detection experiment. The results of the present study demonstrate that body size and colour morph can interactively influence detection of camouflaged prey. This may contribute to the morph frequency differences between male and female pygmy grasshoppers in the wild. Such interactive effects may also influence the dynamics of colour polymorphisms, and contribute to the evolution of ontogenetic colour change and sexual dichromatism. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 112–122.     

Identification of plant cytoskeleton-interacting proteins by screening for actin stress fiber association in mammalian fibroblasts

Taking advantage of the high conservation of the cytoskeleton building blocks actin and tubulin between plant and animal kingdoms, we developed a functional genomic screen for the isolation of new plant cytoskeleton-binding proteins that uses a mammalian cell expression system. A yellow fluorescent protein (YFP)-fusion cDNA library from Arabidopsis was inserted into rat fibroblasts and screened for fluorescent chimeras localizing to cytoskeletal structures. The high-throughput screen was performed by an automated microscope. An initial set of candidate genes identified in the screen was isolated, sequenced, the full-length cDNAs were synthesized by RT-PCR and tested by biochemical approaches to verify the ability of the genes to bind actin directly. Alternatively, indirect binding via interaction with other actin-binding proteins was studied. The full-length cDNAs were transferred back to plants as YFP chimeras behind the CAMV-35S promoter. We give here two examples of new plant cytoskeletal proteins identified in the pilot screen. ERD10, a member of the dehydrin family of proteins, was localized to actin stress fibers in rat fibroblasts. Its direct binding to actin filaments was confirmed by several biochemical approaches. Touch-induced calmodulin-like protein, TCH2, was also localized to actin stress fibers in fibroblasts, but was unable to bind actin filaments directly in vitro. Nevertheless, it did bind to the IQ domains of Arabidopsis myosin VIII in a calcium-dependent manner. Further evidence for a cytoskeletal function of ERD10 was obtained in planta; GFP-ERD10 was able to protect the actin cytoskeleton from latrunculin-mediated disruption in Nicotiana benthamiana leaves.     

Resonance Raman spectra of cytochrome oxidase. Evidence for photoreduction by laser photons in resonance with the Soret band

Resonance Raman spectra of cytochrome oxidase solubilized in Tween 20 and sodium cholate, and excited at 413.1 nm have been recorded. Differences in the resonance Raman spectra of the two preparations are minimal indicating that the local environment of the hemes is similar in the two preparations. As in the work of Salmeen, et al. (1973) (Biochem. Biophys. Res. Commun. 52, 1100) the strongest band appears at 1358 cm^?1. Some of the other bands differ slightly in their band shapes and frequencies when compared to their spectra; these differences can be accounted for by differences in resonance enhancement of the various bands when exciting at 441.6 and 413.1 nm. A study of the region from 1350 to 1380 cm^?1 as a function of laser intensity (10–130 mW on sample) indicate that the doublet reported by Salmeen, et al. at 1358 and 1372 cm^?1 is a result of photoreduction of the preparations. In samples to which potassium ferricyanide had been added, broad luminescence bands appear at 476 and 641 nm from which it is inferred that catalytic amounts of flavin in the preparations are photoreduced providing reducing equivalents to cytochrome oxidase.