The development of chromatography for the characterization of protein interactions: a personal perspective

This article reviews the progress of a personal endeavour to develop chromatography as a quantitative procedure for the determination of reaction stoichiometries and equilibrium constants governing protein interactions. As well as affording insight into an aspect of chromatography with which many protein chemists are unfamiliar, it shows the way in which minor adaptations of conventional chromatographic practices have rendered the technique one of the most powerful methods available for the characterization of interactions. That pathway towards quantification is followed from the introduction of frontal gel filtration for the study of protein self-association to the characterization of ligand binding by the biosensor variant of quantitative affinity chromatography.

Dietary interactions influence the effects of bovine folate-binding protein on the bioavailability of tetrahydrofolates in rats

The newborns of mammals have a high folate demand, yet obtain adequate folate nutrition solely from their mothers' milk despite its low folate content. Milk folate is entirely bound by an excess of folate-binding protein (FBP), prompting speculation that FBP may affect the bioavailability of the limited folate supply. Previous research has shown that FBP-bound folic acid is more gradually absorbed, thereby reducing the peak plasma folate concentration and preventing loss into the urine. Natural folates are reduced derivatives of folic acid, with milk predominantly containing 5-methyltetrahydrofolate, yet little research has been carried out to determine the role of FBP in the bioavailability of reduced folates. We studied the effect of FBP on folate nutrition of rats in both single-dose and 4-wk feeding experiments. The effect of FBP was influenced by the presence of other milk components. FBP increased bioavailability of dietary folate when it was consumed with other whey proteins or with soluble casein. However, in the presence of acid-precipitated casein or a whey preparation enriched in lipids, bioavailability was decreased. These results highlight the difficulties of extrapolating from experimental results obtained using purified diets alone and of studying interactions among dietary components. They suggest that the addition of FBP-rich foods to folate-rich foods could enhance the bioavailability of natural folates, but that the outcome of such a combination would depend on interactions with other components of the diet.

Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

Conformational changes in SP-B as a function of surface pressure

X-ray reflectivity of bovine and sheep surfactant-associated protein B (SP-B) monolayers is used in conjunction with pressure-area isotherms and protein models to suggest that the protein undergoes changes in its tertiary structure at the air/water interface under the influence of surface pressure, indicating the likely importance of such changes to the phenomena of protein squeeze out as well as lipid exchange between the air-water interface and subphase structures. We describe an algorithm based on the well-established box- or layer-models that greatly assists the fitting of such unknown scattering-length density profiles, and which takes the available instrumental resolution into account. Scattering-length density profiles from neutron reflectivity of bovine SP-B monolayers on aqueous subphases are shown to be consistent with the exchange of a large number of labile protons as well as the inclusion of a significant amount of water, which is partly squeezed out of the protein monolayer at elevated surface pressures.

Generation of aldehyde-derived protein modifications in ethanol-exposed heart

Background: Although excessive ethanol consumption is known to lead to a variety of adverse effects in the heart, the molecular mechanisms of such effects have remained poorly defined. We hypothesized that posttranslational covalent binding of reactive molecular species to proteins occurs in the heart in response to acute ethanol exposure. Methods: The generation of protein adducts with several aldehydic species was examined by using monospecific antibodies against adducts with malondialdehyde (MDA), acetaldehyde (AA), MDA-AA hybrids, and hydroxyethyl radicals. Specimens of heart tissue were obtained from rats after intraperitoneal injections with alcohol (75 mmol/kg body weight) with or without pretreatment with cyanamide (0.05 mmol/kg body weight), an aldehyde dehydrogenase inhibitor. Results: The amounts of MDA and unreduced AA adducts were found to be significantly increased in the heart of the rats treated with ethanol, cyanamide, or both, whereas no other adducts were detected in statistically significant quantities. Immunohistochemical studies for characterization of adduct distribution revealed sarcolemmal adducts of both MDA and AA in the rats treated with ethanol and cyanamide in addition to intracellular adducts, which were also present in the group treated with ethanol alone. Conclusions: These findings support the role of enhanced lipid peroxidation and the generation of protein-aldehyde condensates in vivo as a result of excessive ethanol intake. These findings may have implications in the molecular mechanisms of cardiac dysfunction in alcoholics.

Protein film voltammetry of Rhodobacter capsulatus xanthine dehydrogenase

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD(+) as the electron acceptor. R. capsulatus XDH forms an (alphabeta)(2) heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds; however, R. capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. Here we demonstrate electrocatalytic activity of the recombinant enzyme, expressed in Escherichia coli, while immobilized on an edge plane pyrolytic graphite working electrode. Furthermore, we have determined all redox potentials of the four cofactors (Mo-VI/V, Mo-V/IV, FAD/FADH, FADH/FADH(2) and two distinct [2Fe-2S](2+/+) clusters) using a combination of potentiometric and voltammetric methods. A novel feature identified in catalytic voltammetry of XDH concerns the potential for the onset of catalysis (ca. 400 mV), which is at least 600 mV more positive than that of the highest potential cofactor. This unusual observation is explained on the basis of a pterin-associated oxidative switch during voltammetry that precedes catalysis.

Decreased coumarin 7-hydroxylase activities and CYP2A6 expression levels in humans caused by genetic polymorphism in CYP2A6 promoter region (CYP2A6*9)

One of seven poor metabolizers of coumarin found in Thai subjects was previously genotyped as heterozygote for the CYP2A6*4 (whole deletion) and CYP2A6*9. Thus, we aimed to investigate the relationship between the genetic polymorphism in the TATA box of the CYP2A6 gene (CYP2A6*9), expression levels of CYP2A6 mRNA and coumarin 7-hydroxylase activities in human livers. Levels of CYP2A6 mRNA were quantified by real-time quantitative reverse transcriptase-polymerase chain reaction. The mean expression levels of CYP2A6 mRNA in individuals with CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 58%, 71% and 21% of the individuals genotyped as CYP2A6*1/*1, respectively. The mean in-vitro coumarin 7-hydroxylase activities in subjects carrying CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 41%, 71% and 12%, respectively, compared to those of the subjects judged as wild-type. Vmax values for coumarin 7-hydroxylation in the liver microsomes from human subjects with genotypes of CYP2A6*1/*1, CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 0.58, 0.26, 0.44 and 0.13 nmol/min/nmol total P450, respectively. CYP2A6 protein levels in human liver microsomes with the CYP2A6*4 and the CYP2A6*9 alleles were markedly decreased. These results suggest that the genetic polymorphism in the promoter region of the CYP2A6 gene (CYP2A6*9) reduced the expression levels of CYP2A6 mRNA and protein in human livers, resulting in the decrease of coumarin 7-hydroxylase activities. Individuals judged as CYP2A6*4/*9 were expected to be poor metabolizers, having extremely low activity of CYP2A6.

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental antoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP. (C) 2003 Elsevier B.V. All rights reserved.

Dehydration converts DsbG crystal diffraction from low to high resolution

Diffraction quality crystals are essential for crystallographic studies of protein structure, and the production of poorly diffracting crystals is often regarded as a dead end in the process. Here we show a dramatic improvement of poorly diffracting DsbG crystals allowing high-resolution diffraction data measurement. Before dehydration, the crystals are fragile and the diffraction pattern is streaky, extending to 10 Angstrom resolution. After dehydration, there is a spectacular improvement, with the diffraction pattern extending to 2 Angstrom resolution. This and other recent results show that dehydration is a simple, rapid, and inexpensive approach to convert poor quality crystals into diffraction quality crystals.

Sensitivity of Helicoverpa armigera nucleopolyhedrovirus polyhedra to sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) is commonly used to extract polyhedra from infected cells and diseased dead larval tissues. It was found, however, that 80% of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) polyhedra produced via cell culture were damaged after 30 min of 0.5% SDS treatment whereas only 20% of in vivo produced polyhedra were damaged by the same treatment. Transmission and scanning electron microscopy revealed that the damaged polyhedra had lost their polyhedron envelopes and virions were dislodged from the polyhedrin matrix, leaving empty spaces that were previously occupied by the occluded virions. Up to 20% in vitro produced polyhedra were resistant to SDS and remained intact, even after a 24 h exposure to SDS. This sensitivity to SDS was observed across a range of cell culture media, including serum supplemented media. Electron microscopy also revealed that the inferior polyhedron envelope of in vitro produced polyhedra is likely due to poor interaction between the polyhedron envelope, polyhedron envelope protein (PEP), and polyhedrin matrix. The PEP gene was cloned and sequenced and mutations in this gene were ruled out as an explanation. In vitro produced polyhedra that were passed through insect larva once were resistant to SDS, indicating that a critical component is lacking in insect cell culture medium used for producing HaSNPV or the cells growing in culture are inefficient in some ways in relation to production of polyhedra. (C) 2002 Elsevier Science (USA). All rights reserved.

An image processing application for quantification of protein aggregates in Caenorhabditis elegans

Protein aggregation became a widely accepted marker of many polyQ disorders, including Machado-Joseph disease (MJD), and is often used as readout for disease progression and development of therapeutic strategies. The lack of good platforms to rapidly quantify protein aggregates in a wide range of disease animal models prompted us to generate a novel image processing application that automatically identifies and quantifies the aggregates in a standardized and operator-independent manner. We propose here a novel image processing tool to quantify the protein aggregates in a Caenorhabditis elegans (C. elegans) model of MJD. Confocal mi-croscopy images were obtained from animals of different genetic conditions. The image processing application was developed using MeVisLab as a platform to pro-cess, analyse and visualize the images obtained from those animals. All segmenta-tion algorithms were based on intensity pixel levels.The quantification of area or numbers of aggregates per total body area, as well as the number of aggregates per animal were shown to be reliable and reproducible measures of protein aggrega-tion in C. elegans. The results obtained were consistent with the levels of aggrega-tion observed in the images. In conclusion, this novel imaging processing applica-tion allows the non-biased, reliable and high throughput quantification of protein aggregates in a C. elegans model of MJD, which may contribute to a significant improvement on the prognosis of treatment effectiveness for this group of disor-ders

Automatic segmentation and 3D feature extraction of protein aggregates in Caenorhabditis Elegans

In the last years, it has become increasingly clear that neurodegenerative diseases involve protein aggregation, a process often used as disease progression readout and to develop therapeutic strategies. This work presents an image processing tool to automatic segment, classify and quantify these aggregates and the whole 3D body of the nematode Caenorhabditis Elegans. A total of 150 data set images, containing different slices, were captured with a confocal microscope from animals of distinct genetic conditions. Because of the animals’ transparency, most of the slices pixels appeared dark, hampering their body volume direct reconstruction. Therefore, for each data set, all slices were stacked in one single 2D image in order to determine a volume approximation. The gradient of this image was input to an anisotropic diffusion algorithm that uses the Tukey’s biweight as edge-stopping function. The image histogram median of this outcome was used to dynamically determine a thresholding level, which allows the determination of a smoothed exterior contour of the worm and the medial axis of the worm body from thinning its skeleton. Based on this exterior contour diameter and the medial animal axis, random 3D points were then calculated to produce a volume mesh approximation. The protein aggregations were subsequently segmented based on an iso-value and blended with the resulting volume mesh. The results obtained were consistent with qualitative observations in literature, allowing non-biased, reliable and high throughput protein aggregates quantification. This may lead to a significant improvement on neurodegenerative diseases treatment planning and interventions prevention