Isolation and characterization of novel cyclotides from Viola hederaceae - Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide

Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 ( vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H-1 NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded β- sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and ∼ 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 μ m.

Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by H-1 NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 Angstrom, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H-alpha protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.

SCORPION2: A database for structure-function analysis of scorpion toxins

Scorpion toxins are important experimental tools for characterization of vast array of ion channels and serve as scaffolds for drug design. General public database entries contain limited annotation whereby rich structure-function information from mutation studies is typically not available. SCORPION2 contains more than 800 records of native and mutant toxin sequences enriched with binding affinity and toxicity information, 624 three-dimensional structures and some 500 references. SCORPION2 has a set of search and prediction tools that allow users to extract and perform specific queries: text searches of scorpion toxin records, sequence similarity search, extraction of sequences, visualization of scorpion toxin structures, analysis of toxic activity, and functional annotation of previously uncharacterized scorpion toxins. The SCORPION2 database is available at http://sdmc.i2r.a-star.edu.sg/scorpion/. (c) 2006 Elsevier Ltd. All rights reserved.

Surfactant-free purification of membrane proteins with intact native membrane environment

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.

S12C water control structure near SRS-1a, Shark River Slough

This material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DBI-0620409 and #DEB-9910514. This image is made available for non-commercial or educational use only.

Aerial photo of SRS-1a, the S12c water control structure, and the surrounding area, Shark River Slough

This material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DBI-0620409 and #DEB-9910514. This image is made available for non-commercial or educational use only.

Aerial photo of SRS-1a, the S12c water control structure, and the surrounding area, Shark River Slough

This material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DBI-0620409 and #DEB-9910514. This image is made available for non-commercial or educational use only.

Aerial photo of SRS-1a, the S12c water control structure, and the surrounding area, Shark River Slough

This material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DBI-0620409 and #DEB-9910514. This image is made available for non-commercial or educational use only.

Aerial photo of SRS-1a, the S12c water control structure, and the surrounding area, Shark River Slough

This material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DBI-0620409 and #DEB-9910514. This image is made available for non-commercial or educational use only.

Growth and gas exchange responses of Brazilian pepper (Schinus terebinthifolius) and native South Florida species to salinity

Schinus terebinthifolius Raddi (Schinus) is an invasive exotic species widely found in disturbed and native communities of Florida. This species has been shown to displace native species as well as alter community structure and function. The purpose of this study was to determine if the growth and gas exchange patterns of Schinus, under differing salinity conditions, were different from native species. Two native upland glycophytic species (Rapanea punctata and Randia aculeata) and two native mangrove species (Rhizophora mangle and Laguncularia racemosa) were compared with the exotic. Overall, the exotics morphologic changes and gas exchange patterns were most similar to R. mangle. Across treatments, increasing salinity decreased relative growth rate (RGR), leaf area ratio (LAR) and specific leaf area (SLA) but did not affect root/shoot ratios (R:S). Allocation patterns were however significantly different among species. The largest proportion of Schinus biomass was allocated to stems (47%), resulting in plants that were generally taller than the other species. Schinus also had the highest SLA and largest total leaf area of all species. This meant that the exotic, which was taller and had thinner leaves, was potentially able to maintain photosynthetic area comparable to native species. Schinus response patterns show that this exotic exhibits some physiological tolerance for saline conditions. Coupled with its biomass allocation patterns (more stem biomass and large area of thin leaves), the growth traits of this exotic potentially provide this species an advantage over native plants in terms of light acquisition in a brackish forested ecosystem.

Using Florida Keys Reference Sites As a Standard for Restoration of Forest Structure in Everglades Tree Islands

In south Florida, tropical hardwood forests (hammocks) occur in Everglades tree islands and as more extensive forests in coastal settings in the nearby Florida Keys. Keys hammocks have been less disturbed by humans, and many qualify as “old-growth,” while Everglades hammocks have received much heavier use. With improvement of tree island condition an important element in Everglades restoration efforts, we examined stand structure in 23 Keys hammocks and 69 Everglades tree islands. Based on Stand Density Index and tree diameter distributions, many Everglades hammocks were characterized by low stocking and under-representation in the smaller size classes. In contrast, most Keys forests had the dense canopies and open understories usually associated with old-growth hardwood hammocks. Subject to the same caveats that apply to off-site references elsewhere, structural information from mature Keys hammocks can be helpful in planning and implementing forest restoration in Everglades tree islands. In many of these islands, such restoration might involve supplementing tree stocking by planting native trees to produce more complete site utilization and a more open understory.

Antipredator Responses by Native Mosquitofish to Non-Native Cichlids: An Examination of the Role of Prey Naiveté

The strong impact of non-native predators in aquatic systems is thought to relate to the evolutionary naiveté of prey. Due to isolation and limited dispersal, this naiveté may be relatively high in freshwater systems. In this study, we tested this notion by examining the antipredator response of native mosquitofish, Gambusia holbrooki, to two non-native predators found in the Everglades, the African jewelfish, Hemichromis letourneuxi, and the Mayan cichlid, Cichlasoma urophthalmus. We manipulated prey naiveté by using two mosquitofish populations that varied in their experience with the recent invader, the African jewelfish, but had similar levels of experience with the longer-established Mayan cichlid. Specifically, we tested these predictions: (1) predator hunting modes differed between the two predators, (2) predation rates would be higher by the novel jewelfish predator, (3) particularly on the naive population living where jewelfish have not invaded yet, (4) antipredator responses would be stronger to Mayan cichlids due to greater experience and weaker and/or ineffective to jewelfish, and (5) especially weakest by the naive population. We assayed prey and predator behavior, and prey mortality in lab aquaria where both predators and prey were free-ranging. Predator hunting modes and habitat domains differed, with jewelfish being more active search predators that used slightly higher parts of the water column and less of the habitat structure relative to Mayan cichlids. In disagreement with our predictions, predation rates were similar between the two predators, antipredator responses were stronger to African jewelfish (except for predator inspections), and there was no difference in response between jewelfish-savvy and jewelfish-naive populations. These results suggest that despite the novelty of introduced predators, prey may be able to respond appropriately if non-native predator archetypes are similar enough to those of native predators, if prey rely on general antipredator responses or predation cues, and/or show neophobic responses.

Impacts of newly established non-indigenous green crab (Carcinus maenas) on native fauna in Placentia Bay, Newfoundland

The recent invasion of the European green crab (Carcinus maenas) populations in Placentia Bay, Newfoundland and Labrador (NL) raises great concern about potential impacts on local fisheries and native biodiversity. Green crab are highly adaptable and in both native and invaded areas, green crab are well established predators that can outcompete other similarly sized decapods. The main objectives of this thesis were to: 1) identify the native species that green crab compete with for resources; 2) determine the depths and substrate types in which these interactions likely occur; 3) assess the indirect effects of green crab on native crustaceans and their changes in behavior; 4) assess the impacts of green crab on benthic community structure; 5) compare the NL population with other Atlantic Canadian populations in terms of competitive abilities; and 6) compare morphological features of the NL population with other Atlantic Canadian populations. I found that green crab overlap in space and diet with both rock crab (Cancer irroratus) and American lobster (Homarus americanus), potentially leading to a shift in habitat. Laboratory studies on naïve juvenile lobster also suggested shifts in behavior related to green crab, in that lobster decreased foraging activity and increased shelter use in the presence of green crab. Benthic community analyses showed fewer species in mud, sand, and eelgrass sites heavily populated by green crab compared to sites without green crab, although results depended on the taxa involved and I could not eliminate environmental differences through a short term caging study. Foraging ability of green crab varied in intraspecific competition experiments, with populations from NL and Prince Edward Island dominating longer-established populations from Nova Scotia and New Brunswick. Additional studies excluded claw size as a factor driving these results and behavioral differences likely reflected differences in invasion time and population genetics. Overall, green crab in Placentia Bay appear to be altering community structure of benthic invertebrates through predation and they also appear to indirectly impact native crustaceans through competition.