Experimental mapping of soluble protein domains using a hierarchical approach

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained

Pampas deer, armadillos and coypus: autochthonous mammals and land use changes, agricultural managements and presence of new elements in the rural landscape

La actividad agropecuaria ha sido, y continúa siendo, una gigantesca fuerza transformadora denuestro planeta. Los agroecosistemas de las pampas, en Argentina, no han sido la excepción a estas transformaciones:a la expansión agrícola y la masiva adopción del sistema de siembra directa, se le ha sumado elreordenamiento territorial y la intensificación de la ganadería. A su vez, obras de infraestructura (caminos, canalizaciones, terraplenes) impusieron nuevas configuraciones al paisaje rural ¿Cómo ha respondido la mastofaunaa estos cambios? En este trabajo sintetizamos los resultados registrados, entre los cuales podemos señalar: 1) unmarcado uso diferencial de lotes por parte de las especies de armadillos más comunes, con mayor actividad depeludos (Chaetophractus villosus) en rastrojos agrícolas y de mulitas (Dasypus hybridus) en campos ganaderos,2) que la coexistencia entre venados de las pampas (Ozotoceros bezoarticus) y ganado vacuno solo es posiblebajo sistemas de pastoreo que ofrezcan períodos de descanso a los potreros o bajo esquemas de baja cargaganadera, y 3) que los coipos (Myocastor coypus) utilizan diferencialmente los canales artificiales en períodosmás secos. Se discuten las oportunidades y desafíos que estas respuestas tienen para la conservación de especiesamenazadas o el manejo de especies potencialmente conflictivas, y se señalan algunas necesidades futuras deinvestigación en función de los cambios que continúan operando sobre estos sistemas.Agriculture has been and still remains as a huge transforming power of our planet. In Argentina, agroecosystems of the pampas have also changed accordingly, led by agricultural expansion and the massive adoption of no-till system, along with territorial reorganization and intensification of livestock production. In turn, infrastructure (roads, channels trenches) imposed new configurations to the rural landscape. How mammals have responded to these changes? Here we summarize the results recorded, among which we note: 1) a marked differential use of plots by the two most common species of armadillos, large hairy armadillos (Chaetophractus villosus) more active in crop stubbles and southern long-nosed armadillos (Dasypus hybridus) more active in paddocks under livestock use, 2) the coexistence of Pampas deer (Ozotoceros bezoarticus) and cattle is only possible under grazing systems that provide resting periods to paddocks or under low stocking schemes, and 3) that coypu (Myocastor coypus) differentially use artificial channels in drier periods. Opportunities and challenges derived from these responses are discussed for the conservation of endangered species or the management of potentially conflicting species, as well as some future research needs are identified in terms of changes that continue to operate on these systems.Centro de Estudios Parasitológicos y de Vectore

Structural insights into Clostridium perfringens delta toxin pore formation

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins

Pampas deer, armadillos and coypus: autochthonous mammals and land use changes, agricultural managements and presence of new elements in the rural landscape

La actividad agropecuaria ha sido, y continúa siendo, una gigantesca fuerza transformadora denuestro planeta. Los agroecosistemas de las pampas, en Argentina, no han sido la excepción a estas transformaciones:a la expansión agrícola y la masiva adopción del sistema de siembra directa, se le ha sumado elreordenamiento territorial y la intensificación de la ganadería. A su vez, obras de infraestructura (caminos, canalizaciones, terraplenes) impusieron nuevas configuraciones al paisaje rural ¿Cómo ha respondido la mastofaunaa estos cambios? En este trabajo sintetizamos los resultados registrados, entre los cuales podemos señalar: 1) unmarcado uso diferencial de lotes por parte de las especies de armadillos más comunes, con mayor actividad depeludos (Chaetophractus villosus) en rastrojos agrícolas y de mulitas (Dasypus hybridus) en campos ganaderos,2) que la coexistencia entre venados de las pampas (Ozotoceros bezoarticus) y ganado vacuno solo es posiblebajo sistemas de pastoreo que ofrezcan períodos de descanso a los potreros o bajo esquemas de baja cargaganadera, y 3) que los coipos (Myocastor coypus) utilizan diferencialmente los canales artificiales en períodosmás secos. Se discuten las oportunidades y desafíos que estas respuestas tienen para la conservación de especiesamenazadas o el manejo de especies potencialmente conflictivas, y se señalan algunas necesidades futuras deinvestigación en función de los cambios que continúan operando sobre estos sistemas.Agriculture has been and still remains as a huge transforming power of our planet. In Argentina, agroecosystems of the pampas have also changed accordingly, led by agricultural expansion and the massive adoption of no-till system, along with territorial reorganization and intensification of livestock production. In turn, infrastructure (roads, channels trenches) imposed new configurations to the rural landscape. How mammals have responded to these changes? Here we summarize the results recorded, among which we note: 1) a marked differential use of plots by the two most common species of armadillos, large hairy armadillos (Chaetophractus villosus) more active in crop stubbles and southern long-nosed armadillos (Dasypus hybridus) more active in paddocks under livestock use, 2) the coexistence of Pampas deer (Ozotoceros bezoarticus) and cattle is only possible under grazing systems that provide resting periods to paddocks or under low stocking schemes, and 3) that coypu (Myocastor coypus) differentially use artificial channels in drier periods. Opportunities and challenges derived from these responses are discussed for the conservation of endangered species or the management of potentially conflicting species, as well as some future research needs are identified in terms of changes that continue to operate on these systems.Centro de Estudios Parasitológicos y de Vectore

New Molecular Reporters for Rapid Protein Folding Assays

The GFP folding reporter assay [1] uses a C-terminal GFP fusion to report on the folding success of upstream fused polypeptides. The GFP folding assay is widely-used for screening protein variants with improved folding and solubility [2]–[8], but truncation artifacts may arise during evolution, i.e. from de novo internal ribosome entry sites [9]. One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants. Circular permutants of fluorescent proteins often misfold and are non-fluorescent, and do not readily tolerate fused polypeptides within the fluorescent protein scaffolding [10]–[12]. To overcome these limitations, and to increase the dynamic range for reporting on protein misfolding, we have created eight GFP insertion reporters with different sensitivities to protein misfolding using chimeras of two previously described GFP variants, the GFP folding reporter [1] and the robustly-folding “superfolder” GFP [13]. We applied this technology to engineer soluble variants of Rv0113, a protein from Mycobacterium tuberculosis initially expressed as inclusion bodies in Escherichia coli. Using GFP insertion reporters with increasing stringency for each cycle of mutagenesis and selection led to a variant that produced large amounts of soluble protein at 37°C in Escherichia coli. The new reporter constructs discriminate against truncation artifacts previously isolated during directed evolution of Rv0113 using the original C-terminal GFP folding reporter. Using GFP insertion reporters with variable stringency should prove useful for engineering protein variants with improved folding and solubility, while reducing the number of artifacts arising from internal cryptic ribosome initiation sites

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient

Fluorescent Protein-Based Methods for On-Plate Screening of Gene Insertion

Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening

Plasma membrane dynamics and tetrameric organisation of ABCG2 transporters in mammalian cells revealed by single particle imaging techniques

ABCG2 is one of three human ATP binding cassette (ABC) transporters involved in the export from cells of a chemically and structurally diverse range of compounds. This multidrug efflux capability, together with a broad tissue distribution in the body, means that ABCG2 exerts a range of effects on normal physiology such as kidney urate transport, as well as contributing towards the pharmacokinetic profiles of many exogenous drugs. The primary sequence of ABCG2 contains only half the number of domains required for a functioning ABC transporter and so it must oligomerise in order to function, yet its oligomeric state in intact cell membranes remains uncharacterized. We have analysed ABCG2 in living cell membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, and stepwise photobleaching to demonstrate a predominantly tetrameric structure for ABCG2 in the presence or absence of transport substrates. These results provide the essential basis for exploring pharmacological manipulation of oligomeric state as a strategy to modulate ABCG2 activity in future selective therapeutics

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

Circular Permutation of Red Fluorescent Proteins

Circular permutation of fluorescent proteins provides a substrate for the design of molecular sensors. Here we describe a systematic exploration of permutation sites for mCherry and mKate using a tandem fusion template approach. Circular permutants retaining more than 60% (mCherry) and 90% (mKate) brightness of the parent molecules are reported, as well as a quantitative evaluation of the fluorescence from neighboring mutations. Truncations of circular permutants indicated essential N- and C- terminal segments and substantial flexibility in the use of these molecules. Structural evaluation of two cp-mKate variants indicated no major conformational changes from the previously reported wild-type structure, and cis conformation of the chromophores. Four cp-mKates were identified with over 80% of native fluorescence, providing important new building blocks for sensor and complementation experiments