Modelling Förster resonance energy transfer (FRET) using anisotropy resolved multi-dimensional emission spectroscopy (ARMES)

BackgroundFörster Resonance Energy Transfer (FRET) is widely used to study the structure and dynamics of biomolecular systems and also causes the non-linear fluorescence response observed in multi-fluorophore proteins. Accurate FRET analysis, in terms of measuring changes in donor and acceptor spectra and energy transfer efficiency is therefore critical.MethodsWe demonstrate a novel quantitative FRET analysis using anisotropy resolved multidimensional emission spectroscopy (ARMES) in a Human Serum Albumin (HSA) and 1,8-anilinonaphathalene sulfonate (ANS) model. ARMES combines 4D measurement of polarized excitation emission matrices (pEEM) with multivariate data analysis to spectrally resolve contributing fluorophores. Multivariate analysis (Parallel Factor, PARAFAC and restricted Tucker3) was used to resolve fluorophore contributions and for modelling the quenching of HSA emission and the HSA-ANS interactions.ResultspEEM spectra were modelled using Tucker3 which accommodates non-linearities introduced by FRET and a priori chemical knowledge was used to optimise the solution, thus resolving three components: HSA emission, ANS emission from indirect FRET excitation, and ANS emission from direct excitation. Perpendicular emission measurements were more sensitive to indirectly excited acceptor emission. PARAFAC modelling of HSA, donor emission, separated ANS FRET interacting (Tryptophan) and non-interacting (Tyrosine) components. This enabled a new way of calculating quenching constants using the multi-dimensional emission of individual donor fluorophores.ConclusionsFRET efficiency could be calculated using the multi-dimensional, resolved emission of the interacting donor fluorophores only which yielded higher ET efficiencies compared to conventional methods.General significanceShows the potential of multidimensional fluorescence measurements and data analysis for more accurate FRET modelling in proteins.     

Effect of enhanced Renilla luciferase and fluorescent protein variants on the Förster distance of Bioluminescence resonance energy transfer (BRET)

Bioluminescence resonance energy transfer (BRET) is an important tool for monitoring macromolecular interactions and is useful as a transduction technique for biosensor development. Förster distance (R₀), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R₀ provides a means of linking measured changes in BRET ratio to a physical dimension scale and allows estimation of the range of distances that can be measured by any donor–acceptor pair. The sensitivity of BRET assays has recently been improved by introduction of new BRET components, RLuc2, RLuc8 and Venus with improved quantum yields, stability and brightness. We determined R₀ for BRET¹ systems incorporating novel RLuc variants RLuc2 or RLuc8, in combination with Venus, as 5.68 or 5.55 nm respectively. These values were approximately 25% higher than the R₀ of the original BRET¹ system. R₀ for BRET² systems combining green fluorescent proteins (GFP²) with RLuc2 or RLuc8 variants was 7.67 or 8.15 nm, i.e. only 2–9% greater than the original BRET² system despite being ～30-fold brighter.     

Some relationships between the process of pair formation and gamete maturation in Harmothoë imbricata (L) (Annelida: Polychaeta)

Male and female Harmothoë imbricata form pairs in preparation for spawning although the individuals are normally mutually antagonistic. This change in behaviour is due to a reduction in aggression shown by the females as they approach maturity. Immature males attempt to mount but are normally repulsed by the aggressive immature females. No obvious behavioural change occurs during the period of final gamete maturation in the male. Pair formation results in the paired female spawning sooner than would an equivalent female kept in isolation. Release of sperm over the newly formed egg mass is ensured by receptors on the ventral cirri which inform the male that the female has spawned.     

Two new species of genus Ateleute Förster (Hymenoptera, Ichneumonidae, Cryptinae) with a key to the Oriental species

Three species of Ateleute Förster 1869 belonging to the tribe Cryptini of the subfamily Cryptinae (Hymenoptera, Ichneumonidae), collected from Jiangxi Province, China, are reported, of which two are new for science: Ateleute ferruginea Sheng, Broad & Sun, sp. n. and AAteleute zixiensis Sheng, Broad & Sun, sp. n. One, AAteleute densistriata (Uchida, 1955), was previously known from China and Japan. A key to the species of genus Ateleute known in the Oriental Region is provided.     

Species of the genus Mastrus Förster (Hymenoptera, Ichneumonidae) of China with descriptions of two new species parasitizing sawflies (Hymenoptera)

Four species of Mastrus Förster, 1869 are reported from China. Two, Mastrus nigrus Sheng & Zeng, sp. n. reared from Arge pullata (Zaddach) and Mastrus rugotergalis Sheng & Zeng, sp. n. reared from Diprion jingyuanensis Xiao & Zhang, are new to science. One, Mastrus deminuens (Hartig, 1838), is a parasitoid of Pachynematus itoi Okutani. A key to species of Mastrus Förster known in China is provided.     

Förster energy-transfer studies between Trp residues of alpha1-acid glycoprotein (orosomucoid) and the glycosylation site of the protein

Energy-transfer studies between Trp residues of alpha(1)-acid glycoprotein and the fluorescent probe Calcofluor White were performed. Calcofluor White interacts with carbohydrate residues of the protein, while the three Trp residues are located at the surface (Trp-160) and in hydrophobic domains of the protein (Trp-25 and Trp-122). Binding of Calcofluor to the protein induces a decrease in the fluorescence intensity of the Trp residues accompanied by an increase of that of Calcofluor White. Efficiency (E) of Trp fluorescence quenching was determined to be equal to 45%, and the F?rster distance R(o), at which the efficiency of energy transfer is 50%, was calculated to be 18.13 A. This low distance and the value of the efficiency clearly indicate that energy transfer between Trp residues and Calcofluor White is weak.     

A new species of genus Syntactus Förster (Hymenoptera, Ichneumonidae, Ctenopelmatinae) with a key to Oriental and Eastern Palearctic species

A new species, Syntactus jiulianicus Sun & Sheng, sp.n. belonging to the tribe Pionini of the subfamily Ctenopelmatinae (Hymenoptera, Ichneumonidae), collected from Jiangxi Province, China, is reported. Illustrations of the new species and Syntactus delusor (Linnaeus, 1758), Syntactus minor (Holmgren, 1857) and Syntactus varius (Holmgren, 1858) are provided. A key to the species of Syntactus known from the Oriental and Eastern Palaearctic Regions is given.     

A review of the genera Gnathochorisis F?rster and Symplecis F?rster of South Korea,with notes on Korean orthocentrines (Hymenoptera,Ichneumonidae, Orthocentrinae)

Two genera of Korean Orthocentrinae, Gnathochorisis and Symplecis, are reviewed, and keys to species of these genera are provided here. Two new species, Gnathochorisisfuscipes Humala & Lee, sp. n. and Gnathochorisiskoreensis Humala & Lee, sp. n. are described from South Korea. The current state of the taxonomy of Eastern Palaearctic orthocentrines is briefly discussed.     

First record of the genus Phradis Förster (Hymenoptera, Ichneumonidae, Tersilochinae) from the Neotropical Region

One new species of the genus Phradis, Phradis peruvianussp. n., from the mountainous part of Peruvian Amazonia, is described and illustrated. This is the first record of the genus from South America and the Neotropical region.     

Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, F?rster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

A new species of the genus Disogmus Förster (Hymenoptera, Proctotrupoidea, Proctotrupidae) from the Eocene Rovno amber

Disogmus rasnitsyni Kolyada & Perkovsky, sp. n. is described from a fossil inclusion of Late Eocene amber (Ukraine). The new species is most similar to Disogmus basalis (Thomson, 1857), in particular, in the shape of the tyloids and the general shortening of the segments of antennae, but distinctly differs from it and the other species of the genus by having tyloids on flagellar segments 2-4 compared to 3-6 and 4-7 in other species.     

A new species of the genus Probles Förster (Hymenoptera: Ichneumonidae: Tersilochinae) from South Korea

The new species Probles (Euporizon) leei Khalaim, Balueva & Kim sp. nov., is described from South Korea. The new species is characterized by a reduction of the groove between glymma and the ventral part of postpetiole, whereas other species of Probles typically have a well-developed groove. This is the most abundant species of Probles in South Korea, where it accounts for over half of all collected specimens of the genus.     

Description of new species of the genus Ctenochira Förster, 1855 (hymenoptera,ichneumonidae: Tryphoninae) from the Eurasian Subarctic subzone and Siberia,with remarks on the Ichneumonid fauna of the Chukchi Peninsula

13 new species of Ctenochira (Hymenoptera, Ichneumonidae, Tryphoninae) from the Eurasian Subarctic subzone and Siberia are described: C. albosignata sp. n., C. anabar sp. n., C. epipleuralis sp. n., C. gracilicornis sp. n., C. hyperborea sp. n., C. kerzhneri sp. n., C. laticauda sp. n., C. minuta sp. n., C. nigronitens sp. n., C. pallistigma sp. n., C. tixi sp. n., C. trochanterata sp. n., and C. uzon sp. n. A key to the Subarctic and northern Siberian species is given. Quantitative occurrence of different ichneumonid subfamilies in the Subarctic subzone is considered based on analysis of 827 specimens of ichneumonids collected in yellow plates in the Chukchi Autonomous Area in July 2012. Idiobiont (polyphagous) subfamilies (36.5%): Cryptinae, 294 spms. (35.5%); Pimplinae, 8 (1%). Koinobiont parasitoids, 10 subfamilies (63.5%): parasitoids of Symphyta, 313 spms. (37.9%): Tryphoninae, 225 (27.2%); Adelognathinae, 14 (1.7%); Ctenopelmatinae, 59 (7.2%); Campopleginae (Olesicampe; Lathrostizus), 15 (1.8%); parasitoids of Diptera, 169 spms. (20.5%): Diplazontinae, 8 (1%); Orthocentrinae s. l., 161 (19.5%); parasitoids of Lepidoptera, 29 spms. (3.5%): Campopleginae (part), 18 (2.2%) (Campoletis; Hyposoter; Tranosema; Diadegma; etc.); Ichneumoninae, 2 (0.2%); Banchinae, 3 (0.4%) (Glypta; Lissonota); Metopiinae, 6 (0.7%) (Exochus); parasitoids of Coleoptera (0.3%): Tersilochinae, 3 spms. (Barycnemis); hyperparasitoids (1.3%): Mesochorinae, 11 spms.     

Description of Aprostocetus anoplophorae n. sp. (Hymenoptera: Eulophidae), a new egg parasitoid of the invasive pest Anoplophora chinensis (Förster) (Coleoptera: Cerambycidae)

Aprostocetus anoplophorae n. sp. (Hymenoptera: Eulophidae) is supposed to play a role as an egg parasitoid of the invasive pest, the Citrus Longhorned Beetle, Anoplophora chinensis (Förster). The studies of its morphology, and rDNA sequence data, strongly indicate that this taxon differs greatly from all described Aprostocetus species, and is new to science. This species is described and illustrated. Both its systematic placement and origin are discussed.     

Review of the ichneumon-flies of the genus <Emphasis Type="Italic">Rhorus</Emphasis> Förster, 869 (Hymenoptera,Ichneumonidae: Ctenopelmatinae): I. The species from the Far East (with description of 24 new species and with a key)

34 Palaearctic species of Rhorus are considered, 9 groups of species are distinguished, and 24 new species from the Eastern Palaearctic (mainly from the Russian Far East) and one new species reared in St. Petersburg from Phyllocolpa leucosticta are described. A key to all these species is given.     

NMR study of short β(1-3)-glucans provides insights into the structure and interaction with Dectin-1

β(1-3)-Glucans, abundant in fungi, have the potential to activate the innate immune response against various pathogens. Although part of the action is exerted through the C-type lectin-like receptor Dectin-1, details of the interaction mechanism with respect to glucan chain-length remain unclear. In this study, we investigated a set of short β(1-3)-glucans with varying degree of polymerization (DP); 3, 6, 7, 16, and laminarin (average DP; 25), analyzing the relationship between the structure and interaction with the C-type lectin-like domain (CTLD) of Dectin-1. The interaction of short β(1-3)-glucans (DP6, DP16, and laminarin) with the CTLD of Dectin-1 was systematically analyzed by ¹H-NMR titration as well as by saturation transfer difference (STD)-NMR. The domain interacted weakly with DP6, moderately with DP16 and strongly with laminarin, the latter plausibly forming oligomeric protein-laminarin complexes. To obtain structural insights of short β(1-3)-glucans, the exchange rates of hydroxy protons were analyzed by deuterium induced ¹³C-NMR isotope shifts. The hydroxy proton at C4 of laminarin has slower exchange with the solvent than those of DP7 and DP16, suggesting that laminarin has a secondary structure. Diffusion ordered spectroscopy revealed that none of the short β(1-3)-glucans including laminarin forms a double or triple helix in water. Insights into the interaction of the short β(1-3)-glucans with Dectin-1 CTLD provide a basis to understand the molecular mechanisms of β-glucan recognition and cellular activation by Dectin-1.     

Palaearctic species of the ichneumon-fly genus Campodorus Förster (Hymenoptera, Ichneumonidae): III. Species with long-haired ovipositor sheath, species with entirely rufous hind tibia, and species with white-banded tibiae

A complex of about 40 Palaearctic species of the genus Campodorus with long haired ovipositor sheath and with entirely rufous or white-banded hind tibia is considered and keyed. Twenty-one new species are described. The species with the pale rufous hind tibia have black hind coxa and trochanters, most of them also have rather long genae and scabrous mesopleura. The majority of these species are distributed in subarctic Siberia: C. yakutator sp. n., C. minutor sp. n., C. arctor sp. n., C. atripes sp. n., C. pequenitor sp. n., C. mordax sp. n., C. lucidator sp. n., C. subarctor sp. n., C. nikandrovskii sp. n., and C. genator sp. n. C. suomi sp. n. is described from northern Finland, C. labytnangi sp. n., from Kola Peninsula, the Polar Ural, and Transbaikalia, and only C. gallicator sp. n., from France (? Alps). The species with a white band on the hind tibia are mostly boreal; most of them have red coxae and trochanters (except for C. sakhalinator sp. n. and C. taigator sp. n.), rather short genae, and granulate mesopleura. In this group, eight new species are described: C. atrofemorator sp. n., C. barbator sp. n., C. sakhalinator sp. n. (all from the southern part of the Russian Far East), C. taigator sp. n. (southern Siberian), C. ciliator sp. n., C. melanopygus sp. n., C. marginator sp. n., and C. boreator sp. n. (European-Siberian).     

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid β peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the amyloid β precursor protein (APP) juxtamembrane region

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.