Identifying regions of membrane proteins in contact with phospholipid head groups: covalent attachment of a new class of aldehyde lipid labels to cytochrome c oxidase

A series of amine-specific reagents based on the benzaldehyde reactive group have been synthesized, characterized, and used to study beef heart cytochrome c oxidase reconstituted in phospholipid bilayers. The series contained three classes of reagents: lipid-soluble phosphodiesters having a single hydrocarbon chain, phospholipid analogues, and a water-soluble benzaldehyde. All reagents were either radiolabeled or spin-labeled or both. The Schiff bases formed by these benzaldehydes with amines were found to be reversible until the addition of the reducing agent sodium cyanoborohydride, whereas attachment of lipid-derived aliphatic aldehydes was not readily reversible in the absence of the reducing agent. The benzaldehyde group provides a convenient method of controlling and delaying permanent attachment to integral membrane proteins until after the reconstitution steps. This ensures that the lipid analogues are located properly to identify amine groups at the lipid-protein interface rather than reacting indiscriminately with amines of the hydrophilic domains of the protein. The benzaldehyde lipid labels attach to cytochrome c oxidase with high efficiency. Typically, 20% of the amount of lipid label present was covalently attached to the protein, and the number of moles of label incorporated per mole of protein ranged from 1 to 6, depending on the molar ratios of label, lipid, and protein. The efficiency of labeling by the water-soluble benzaldehyde was much less than that observed for any of the lipid labels because of dilution effects, but equivalent levels of incorporation were achieved by increasing the label concentration. Electron spin resonance spectra of a nitroxide-containing phospholipid analogue covalently attached to reconstituted cytochrome c oxidase exhibited a large motion-restricted component, which is characteristic of spin-labeled lipids in contact with the hydrophobic surfaces of membrane proteins. The line shape and splittings were similar for covalently attached label and label free to diffuse and contact the protein molecules in the bilayer, providing independent evidence that the coupling occurs at the protein-lipid interface. The distribution of the benzaldehyde reagents attached to the polypeptide components of cytochrome c oxidase was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeling pattern observed for the lipid analogues was not affected by the presence of the nitroxide moiety on the acyl chains but was dependent on the molar ratio of labeling reagent to protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Global domination by crazy ants: phylogenomics reveals biogeographical history and invasive species relationships in the genus Nylanderia (Hymenoptera: Formicidae)

Nylanderia (Emery) is one of the world's most diverse ant genera, with 123 described species worldwide and hundreds more undescribed. Fifteen globetrotting or invasive species have widespread distributions and are often encountered outside their native ranges. A molecular approach to understanding the evolutionary history and to revision of Nylanderia taxonomy is needed because historical efforts based on morphology have proven insufficient to define major lineages and delimit species boundaries, especially where adventive species are concerned. To address these problems, we generated the first genus-wide genomic dataset of Nylanderia using ultraconserved elements (UCEs) to resolve the phylogeny of major lineages, determine the age and origin of the genus, and describe global biogeographical patterns. Sampling from seven biogeographical regions revealed a Southeast Asian origin of Nylanderia in the mid-Eocene and four distinct biogeographical clades in the Nearctic, the Neotropics, the Afrotropics/Malagasy region, and Australasia. The Nearctic and Neotropical clades are distantly related, indicating two separate dispersal events to the Americas between the late Oligocene and early Miocene. We also addressed the problem of misidentification that has characterized species-level taxonomy in Nylanderia as a result of limited morphological variation in the worker caste by evaluating the integrity of species boundaries in six of the most widespread Nylanderia species. We sampled across ranges of species in the N. bourbonica complex (N. bourbonica (Forel) + N. vaga (Forel)), the N. fulva complex (N. fulva (Mayr) + N. pubens (Forel)), and the N. guatemalensis complex (N. guatemalensis (Forel) + N. steinheili (Forel)) to clarify their phylogenetic placement. Deep splits within these complexes suggest that some species names – specifically N. bourbonica and N. guatemalensis – each are applied to multiple cryptic species. In exhaustively sampling Nylanderia diversity in the West Indies, a ‘hot spot’ for invasive taxa, we found five adventive species among 22 in the region; many remain morphologically indistinguishable from one another, despite being distantly related. We stress that overcoming the taxonomic impediment through the use of molecular phylogeny and revisionary study is essential for conservation and invasive species management.     

Interaction of extracellular Pseudomonas lipase with alginate and its potential use in biotechnology

Summary Extracellular Pseudomonas lipase is able to interact directly or indirectly with alginate as deduced from the following results: (i) During adsorption chromatography of exolipase the enzyme adsorbed quantitatively to glass beads in the absence of alginate, but not after its preincubation in the presence of the polysaccharide; pretreatment of glass beads with alginate did not prevent enzyme adsorption. (ii) In the presence of alginate exolipase was much more resistant to heat inactivation than in its absence. (iii) In the presence of alginate the increase in exolipase activity caused by the non-ionic detergent Triton X-100 was drastically reduced. (iv) Exolipase could be rapidly and almost completely harvested from cell-free culture fluid of P. aeruginosa 5940 by ethanolic coprecipitation with alginate. After dissolving the coprecipitate in detergent-containing buffer exolipase and polysaccharide could be easily separated by ion-exchange chromatography on DEAE-Sephadex A-25. The coprecipitation method was also successfully applied to exolipases produced by Pseudomonas sp., Chromobacierium viscosum and Rhizopus delamar, thus suggesting potential use of this method in biotechnology.     

Genic homozygosity in an ancient reptile (Alligator mississippiensis)

Proteins assayed electrophoretically showed variation at only three of 49 presumed genetic loci in alligators from southwestern Louisiana. Average heterozygosity per individual was 0.021±0.012; proportion of polymorphic loci was 0.06. Data on the history, structure, and ecology of this alligator population are consistent with natural selection as the primary factor accounting for this low genetic variability. However, neither a historic population bottleneck nor some genetic mechanism limiting variability can be dismissed as a possible factor.The study was supported by NSF Grant BMS 73-0125 to H.C.D.