Effect of cleavage enzyme, search algorithm and decoy database on mass spectrometric identification of wheat gluten proteins

While tandem mass spectrometry (MS/MS) is routinely used to identify proteins from complex mixtures, certain types of proteins present unique challenges for MS/MS analyses. The major wheat gluten proteins, gliadins and glutenins, are particularly difficult to distinguish by MS/MS. Each of these groups contains many individual proteins with similar sequences that include repetitive motifs rich in proline and glutamine. These proteins have few cleavable tryptic sites, often resulting in only one or two tryptic peptides that may not provide sufficient information for identification. Additionally, there are less than 14,000 complete protein sequences from wheat in the current NCBInr release. In this paper, MS/MS methods were optimized for the identification of the wheat gluten proteins. Chymotrypsin and thermolysin as well as trypsin were used to digest the proteins and the collision energy was adjusted to improve fragmentation of chymotryptic and thermolytic peptides. Specialized databases were constructed that included protein sequences derived from contigs from several assemblies of wheat expressed sequence tags (ESTs), including contigs assembled from ESTs of the cultivar under study. Two different search algorithms were used to interrogate the database and the results were analyzed and displayed using a commercially available software package (Scaffold). We examined the effect of protein database content and size on the false discovery rate. We found that as database size increased above 30,000 sequences there was a decrease in the number of proteins identified. Also, the type of decoy database influenced the number of proteins identified. Using three enzymes, two search algorithms and a specialized database allowed us to greatly increase the number of detected peptides and distinguish proteins within each gluten protein group.     

Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine

We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.     

Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour

Background  

The gamma gliadins are a complex group of proteins that together with other gluten proteins determine the functional properties of wheat flour. The proteins have unusually high levels of glutamine and proline and contain large regions of repetitive sequences. While most gamma gliadins are monomeric proteins containing eight conserved cysteine residues, some contain an additional cysteine residue that enables them to be linked with other gluten proteins into large polymers that are critical for flour quality. The ability to differentiate among the gamma gliadins is important for studies of wheat flour quality because proteins with similar sequences can have different effects on functional properties.     

Structure and function in rhodopsin: mapping light-dependent changes in distance between residue 65 in helix TM1 and residues in the sequence 306-319 at the cytoplasmic end of helix TM7 and in helix H8.

Spin-labeled double mutants of rhodopsin were produced containing a reference nitroxide at position 65, at the cytoplasmic termination of helix TM1, and a second nitroxide in the sequence of residues 306-319, which includes the cytoplasmic termination of helix TM7 and nearly the entire surface helix H8. Magnetic dipole-dipole interactions between the spins are analyzed to provide interspin distance distributions in both the dark and photoactivated states of rhodopsin. The distributions, apparently resulting from the conformational flexibility of the side chains, are found to be consistent with the structural model of rhodopsin in the dark state derived from crystallography. Photoactivation of the receptor triggers an increase in distance between residues in TM7, but not those in H8, relative to the reference at position 65 in TM1. The simplest interpretation of the result is a movement of the cytoplasmic portion of TM7 away from TM1 by 2-4 A.     

A model for the structure of the Escherichia coli SOS-regulated UmuD2 protein

The ubiquitous Y-family of DNA polymerases, exemplified by the Escherichia coli UmuC protein (the catalytic subunit of DNA Pol V), possess the remarkable ability to replicate imperfect DNA templates that cannot be replicated by other types of DNA polymerases. Since this ability comes at the cost of a reduced fidelity, it is important that organisms manage these unique polymerases to coordinate their actions with those of the replication machinery. In E. coli, it is becoming evident that a sophisticated series of protein-protein interactions involving the two forms of the umuD gene product, UmuD and UmuD' and components of the replicative DNA polymerase serve to manage the actions of the umuC-encoded DNA polymerase. The purpose of this study was to better understand how structural differences between UmuD2 and UmuD2' help to determine which biological role the umuDC gene products will play; the UmuD2C complex functions as a DNA damage checkpoint effector, while the UmuD2'C complex participates in translesion DNA synthesis, which serves as the mechanistic basis for most chemical and UV light mutagenesis. Based on the results of a combination of disulfide cross-linking experiments, measurements of solvent accessibility and electron paramagnetic spin resonance (EPR) studies, we have developed a refined model for the structure of the UmuD2 homodimer. In the model that we are proposing, the N-terminal arms of UmuD (residues 1-39) form an extended interface in the UmuD2 homodimer by folding down over the globular domains of their intradimer partners. As a result, significant portions of the surface of each globular domain are buried in the UmuD2 homodimer. Based on the structure of the UmuD2' homodimer, both in the crystal and in solution, these same surfaces are exposed. Implications of these structural differences between the UmuD2 and the UmuD2' homodimers with respect to their roles in managing the actions of the umuC-encoded DNA polymerase are discussed.     

Synthesis, characterization and biological evaluation of some 16E-arylidene androstane derivatives as potential anticancer agents

A series of new 16E-arylidene androstane derivatives were synthesized and characterized. The new compounds were screened for their anticancer activities against the human cancer cell lines SW480, A549, HepG2 and HeLa in vitro using the MTT assay. The results of the in vitro study showed that a number of compounds have shown IC₅₀ values lower than 20 μM against the four cancer cell lines.     

Site-directed spin labeling electron paramagnetic resonance study of the ORF1 protein from a mouse L1 retrotransposon

Long interspersed nuclear element-1 is a highly abundant mammalian retrotransposon that comprises 17% of the human genome. L1 retrotransposition requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. ORF1p has been shown to adopt a homo-trimeric, asymmetric dumbbell-shaped structure. However, its atomic-level structure and mechanism of RNA binding remains poorly understood. Here, we report the results of a site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) study of 27 residues within the RNA binding region of the full-length protein. The EPR data are compatible with the large RNA binding lobe of ORF1p containing a RNA recognition motif (RRM) domain and a carboxyl-terminal domain (CTD) that are predicted from crystallographic and NMR studies of smaller fragments of the protein. Interestingly, the EPR data indicate that residues in strands β3 and β4 of the RRM are structurally unstable, compatible with the previously observed sensitivity of this region to proteolysis. Affinity measurements and RNA-dependent EPR spectral changes map the RNA binding site on ORF1p to residues located in strands β3 and β4 of the RRM domain and to helix α1 of the CTD. Complementary in vivo studies also identify residues within the RRM domain that are required for retrotransposition. We propose that in the context of the full-length trimeric protein these distinct surfaces are positioned adjacent to one another providing a continuous surface that may interact with nucleic acids.     

High Resolution Structure and Double Electron-Electron Resonance of the Zebrafish Voltage-dependent Anion Channel 2 Reveal an Oligomeric Population

In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and -3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the VDAC2 and -3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish VDAC2 (zfVDAC2). Further, we resolved the structure of zfVDAC2 at 2.8 Å resolution, revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of ∼20% in lauryldimethine amine oxide detergent micelles, whereas in lipidic bicelles a higher population of dimeric and higher order oligomers species were observed. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1.     

Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure

Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40–80 °C) maximum in situ temperatures (∼23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40–60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii^T (∼96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii^Tand D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 °C for 15 min; D. kuznetsovii, 135 °C for 15 min, maximum of 154 °C reached) and high-temperature conditions in an oil bath (C1A60, 130° for 30 min, D. kuznetsovii 140 °C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (∼30 × higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.