Investigating Models of Protein Function and Allostery With a Widespread Mutational Analysis of a Light-Activated Protein

To investigate the relationship between a protein’s sequence and its biophysical properties, we studied the effects of more than 100 mutations in Avena sativa light-oxygen-voltage domain 2, a model protein of the Per-Arnt-Sim family. The A. sativa light–oxygen–voltage domain 2 undergoes a photocycle with a conformational change involving the unfolding of the terminal helices. Whereas selection studies typically search for winners in a large population and fail to characterize many sites, we characterized the biophysical consequences of mutations throughout the protein using NMR, circular dichroism, and ultraviolet/visible spectroscopy. Despite our intention to introduce highly disruptive substitutions, most had modest or no effect on function, and many could even be considered to be more photoactive. Substitutions at evolutionarily conserved sites can have minimal effect, whereas those at nonconserved positions can have large effects, contrary to the view that the effects of mutations, especially at conserved positions, are predictable. Using predictive models, we found that the effects of mutations on biophysical function and allostery reflect a complex mixture of multiple characteristics including location, character, electrostatics, and chemistry.     

Impact of an exotic parasitoid on Plutella xylostella (Lepidoptera: Plutellidae) population dynamics, damage and indigenous natural enemies in Kenya

Diadegma semiclausum (Hellén) (Hymenoptera: lchneumonidae), an exotic diamondback moth parasitoid, was released in two pilot areas (Werugha in Coast Region and Tharuni in Central Province) in Kenya. Fifteen month before release, observations on the diamondback moth, Plutella xylostella (Linnaeus), and local natural enemy population dynamics and pest damage were initiated in both areas and continued for three years after release. The P. xylostella population was bimodal with higher records during dry seasons. At Werugha, the peak population of P. xylostella was 16.8 per plant (October 2001); at Tharuni it was 12.8 (February 2002). Populations at Werugha declined from three months after release and decreased from 5.4 per plant (before release) to 0.8 (year 3 after release). Concurrently, average damage (1.9 to 1.5) (on a 0-5 scale), proportion of attacked plants (72 to 31%) and proportion of plants in damage group >2 (plants with head damage) decreased (21.4 to 5.3%), while total parasitism increased from 14.4 (before) to 52.5% (year 3 after release, 90% due to D. semiclausum). At Tharuni, D. semiclausum was only recovered 3 months after release. Average populations of P. xylostella declined from 5.9 per plant (before release) to 2.4 (year 3 after release) and damage scores from 2.3 to 1.7. The proportion of plants in damage group >2 declined from 39.7 to 4.5% while overall parasitism increased from 4.2 to 40.6% (98.3% by D. semiclausum). Four species of indigenous parasitoids (Diadegma mollipla (Holmgren), Oomyzus sokolowskii (Kurdjumov), Apanteles sp. and Itoplectis sp., all primary parasitoids) were almost completely displaced by D. semiclausum. Possible reasons for the different parasitoid development between the two release areas and the displacement of the indigenous species are discussed.     

Assembly and architecture of Escherichia coli divisome proteins FtsA and FtsZ

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.     

The Open Microscopy Environment (OME) Data Model and XML file: open tools for informatics and quantitative analysis in biological imaging

The Open Microscopy Environment (OME) defines a data model and a software implementation to serve as an informatics framework for imaging in biological microscopy experiments, including representation of acquisition parameters, annotations and image analysis results. OME is designed to support high-content cell-based screening as well as traditional image analysis applications. The OME Data Model, expressed in Extensible Markup Language (XML) and realized in a traditional database, is both extensible and self-describing, allowing it to meet emerging imaging and analysis needs.     

Deaths of detainees in the custody of US forces in Iraq and Afghanistan from 2002 to 2005

In light of the large number of detainees who continue to be taken and held in US custody in settings with limited judicial or public oversight, deaths of detainees warrant scrutiny. We have undertaken the task of reviewing all known detainee deaths between 2002 and early 2005 based on reports available in the public domain. Using documents obtained from the Department of Defense through a Freedom of Information Act request, combined with a review of anecdotal published press accounts, 112 cases of death of detainees in United States custody (105 in Iraq, 7 in Afghanistan) during the period from 2002 to early 2005 were identified. Homicide accounted for the largest number of deaths (43) followed by enemy mortar attacks against the detention facility (36). Deaths attributed to natural causes numbered 20. Nine were listed as unknown cause of death, and 4 were reported as accidental or natural. A clustering of 8 deaths ascribed to natural causes in Iraq in August 2003 raises questions about the adequacy and availability of medical care, as well as other conditions of confinement that may have had an impact on the mortality rate.     

Heterologous expression of the benzoate para-hydroxylase encoding gene (CYP53B1) from Rhodotorula minuta by Yarrowia lipolytica

There is currently an increasing number of cytochrome P450 (CYP450) monooxygenase encoding genes becoming available from various genome-sequencing projects. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. In this study, the CYP53B1 gene, which encodes a benzoate para-hydroxylase, was successfully cloned from Rhodotorula minuta and overexpressed in Yarrowia lipolytica E150. Multiple copies of the CYP53B1 cDNA were cloned under the POX2 promoter, while the Y. lipolytica CPR was cloned under the isocitrate lyase promoter. Whole cell biotransformation of benzoic acid to para-hydroxybenzoic acid (pHBA) was used to analyse the hydroxylase activity of the recombinant Y. lipolytica UOFS Y-2366. Different induction conditions were tested in shake flask cultures. The highest concentration of pHBA produced by UOFS Y-2366 was 1.6 g l⁻¹ after 200 h when stearic acid was repeatedly added to the media. R. minuta accumulated up to 1.8 g l⁻¹ of pHBA within only 24 h. Thus, the specific hydroxylase activity of Y. lipolytica UOFS Y-2366 [approximately 0.07 U (g dry wt.)⁻¹] was about 30 times lower than the specific hydroxylase activity of R. minuta [2.62 U (g dry wt.)⁻¹]. However, the hydroxylation activity obtained with Y. lipolytica was one of the highest hydroxylation activities thus reported for whole cell biotransformation studies carried out with yeasts expressing foreign CYP450s.     

Quality assessment and analysis of Biogen Idec compound library

A subset of the compound repository for lead identification at Biogen Idec was characterized for its chemical stability over a 3-year period. Compounds were stored at 4 degrees C as 10 mM DMSO stocks, and a small subset of compounds was stored as lyophilized dry films. Compound integrity of 470 discrete compounds (Compound Set I) and 1917 combinatorial chemistry-derived compounds (Compound Set II) was evaluated by liquid chromatography/mass spectrometry from the time of acquisition into the library collection and after 3 years of storage. Loss of compound integrity over the 3 years of storage was observed across the 2 subsets tested. Of Compound Set I, 63% of samples retained > 80% purity, whereas 57% of samples from Compound Set II had purity greater than 60%. The stability of the lyophilized samples was superior to the samples stored as DMSO solution. Although storage at 4 degrees C as DMSO solution was adequate for the majority of compounds, the authors observed and quantified the level of degradation within the compound collection. Their study provides general insight into compound storage and selection of library subsets for future lead identification activities.     

TULIPs: tunable, light-controlled interacting protein tags for cell biology

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.     

CENP-V is required for centromere organization, chromosome alignment and cytokinesis

The mechanism of mitotic chromosome condensation is poorly understood, but even less is known about the mechanism of formation of the primary constriction, or centromere. A proteomic analysis of mitotic chromosome scaffolds led to the identification of CENP-V, a novel kinetochore protein related to a bacterial enzyme that detoxifies formaldehyde, a by-product of histone demethylation in eukaryotic cells. Overexpression of CENP-V leads to hypercondensation of pericentromeric heterochromatin, a phenotype that is abolished by mutations in the putative catalytic site. CENP-V depletion in HeLa cells leads to abnormal expansion of the primary constriction of mitotic chromosomes, mislocalization and destabilization of the chromosomal passenger complex (CPC) and alterations in the distribution of H3K9me3 in interphase nucleoplasm. CENP-V-depleted cells suffer defects in chromosome alignment in metaphase, lagging chromosomes in anaphase, failure of cytokinesis and rapid cell death. CENP-V provides a novel link between centromeric chromatin, the primary constriction and the CPC.     

FAT AND CARBOHYDRATE METABOLISM IN HUMANS—A Study of Nutritional and Hormonal Effects

As an index to the rate of fat utilization in human subjects, the recovery of all radioactive carbon dioxide in the expired air was measured for one hour following intravenous injection of palmitate-1-C¹⁴. In the normal fasted subject, about 10 per cent of the injected dose was recovered, and the proportion was lowered to about 5 per cent by administration of glucose. With prolonged fasting, the recovery of radioactive carbon dioxide did not change, despite a rising concentration of fatty acids in the serum. This was interpreted as due to the development of a balance between increasing mobilization and oxidation and was thought to indicate increasing fatty acid oxidation.In chronic undernutrition and diabetes mellitus there was increased fatty acid oxidation due presumably to adaptation to a chronic increase in fat utilization for energy.Administration of human growth hormone did not increase fat oxidation but prevented the usual inhibition produced by glucose. This was interpreted to mean that growth hormone increases fat utilization only indirectly by inhibiting the usual preferential utilization of glucose over fat.