Characterization of pertussis toxin by LC-MS/MS

Liquid chromatography-mass spectrometry (LC-MS) with a dual spray electrospray ionization source has been used to measure the molecular weights of pertussis toxin (PT) subunits. Measurement accuracy better than 0.4 Da was achieved for all PT subunits in the molecular weight range of 11,000 to 27,000 Da. At this mass assignment accuracy level, the sequences of the PT subunits investigated in this study are easily determined based on molecular weight alone. The subunits 1, 2, and 5 of PT were observed to undergo oxidation under normal storage conditions as ammonium sulfate suspension at 2 to 8 degrees C. These oxidized subunits can be separated completely or partially by reverse-phase high-performance liquid chromatography (HPLC) from their native counterparts. For the determination of oxidation sites, the oxidized subunits and their nonoxidized counterparts were fraction collected, trypsin digested, and mapped by LC-MS. The oxidized peptides and their nonoxidized counterparts were further studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to confirm their identities. The methionines at position 212 of subunit 1, at position 89 of subunit 2, and at position 40 of subunit 5 were found to be the primary sites of oxidation.     

Primary Cilia Mediate PGE₂ Release in MC3T3-E1 Osteoblasts

This article has no abstract.     

Structure of the Bacterial Deacetylase LpxC Bound to the Nucleotide Reaction Product Reveals Mechanisms of Oxyanion Stabilization and Proton Transfer

The emergence of antibiotic-resistant strains of pathogenic bacteria is an increasing threat to global health that underscores an urgent need for an expanded antibacterial armamentarium. Gram-negative bacteria, such as Escherichia coli, have become increasingly important clinical pathogens with limited treatment options. This is due in part to their lipopolysaccharide (LPS) outer membrane components, which dually serve as endotoxins while also protecting Gram-negative bacteria from antibiotic entry. The LpxC enzyme catalyzes the committed step of LPS biosynthesis, making LpxC a promising target for new antibacterials. Here, we present the first structure of an LpxC enzyme in complex with the deacetylation reaction product, UDP-(3-O-(R-3-hydroxymyristoyl))-glucosamine. These studies provide valuable insight into recognition of substrates and products by LpxC and a platform for structure-guided drug discovery of broad spectrum Gram-negative antibiotics.     

Kir2.4 Surface Expression and Basal Current Are Affected by Heterotrimeric G-Proteins

Kir2.4, a strongly rectifying potassium channel that is localized to neurons and is especially abundant in retina, was fished with yeast two-hybrid screen using a constitutively active Gα_o1. Here, we wished to determine whether and how Gα_o affects this channel. Using transfected HEK 293 cells and retinal tissue, we showed that Kir2.4 interacts with Gα_o, and this interaction is stronger with the GDP-bound form of Gα_o. Using two-electrode voltage clamp, we recorded from oocytes that were injected with Kir2.4 mRNA and a combination of G-protein subunit mRNAs. We found that the wild type and the inactive mutant of Gα_o reduce the Kir2.4 basal current, whereas the active mutant has little effect. Other pertussis-sensitive Gα subunits also reduce this current, whereas Gα_s increases it. Gβγ increases the current, whereas m-phosducin, which binds Gβγ without affecting the state of Gα, reduces it. We then tested the effect of G-protein subunits on the surface expression of the channel fused to cerulean by imaging the plasma membranes of the oocytes. We found that the surface expression is affected, with effects paralleling those seen with the basal current. This suggests that the observed effects on the current are mainly indirect and are due to surface expression. Similar results were obtained in transfected HEK cells. Moreover, we show that in retinal ON bipolar cells lacking Gβ3, localization of Kir2.4 in the dendritic tips is reduced. We conclude that Gβγ targets Kir2.4 to the plasma membrane, and Gα_o slows this down by binding Gβγ.     

Studying the S-nitrosylation of model peptides and eNOS protein by mass spectrometry.

Oxidative addition of a nitric oxide (NO) molecule to the thiol group of cysteine residues is a physiologically important post-translational modification that has been implicated in several metabolic and pathophysiological events. Our previous studies have indicated that S-nitrosylation can result in the disruption of the endothelial NO synthase (eNOS) dimer. It has been suggested that for S-nitrosylation to occur, the cysteine residue must be flanked by hydrophilic residues either in the primary structure or in the spatial proximity through appropriate conformation. However, this hypothesis has not been confirmed. Thus, the objective of this study was to determine if the nature of the amino acid residues that flank the cysteine in the primary structure has a significant effect on the rate and/or specificity of S-nitrosylation. To accomplish this, we utilized several model peptides based on the eNOS protein sequence. Some of these peptides contained point mutations to allow for different combinations of amino acid properties (acidic, basic, and hydrophobic) around the cysteine residue. To ensure that the results obtained were not dependent on the nitrosylation procedure, several common S-nitrosylation techniques were used and S-nitrosylation followed by mass spectrometric detection. Our data indicated that all peptides independent of the amino acids surrounding the cysteine residue underwent rapid S-nitrosylation. Thus, there does not appear to be a profound effect of the primary sequence of adjacent amino acid residues on the rate of cysteine S-nitrosylation at least at the peptide levels. Finally, our studies using recombinant human eNOS confirm that Cys98 undergoes S-nitrosylation. Thus, our data validate the importance of Cys98 in regulating eNOS dimerization and activity, and the utility of mass spectroscopy to identify cysteine residues susceptible to S-nitrosoylation.     

Charge‐Transfer States at Organic–Organic Interfaces: Impact of Static and Dynamic Disorders

Molecular dynamics simulations are combined with density functional theory calculations to evaluate the impact of static and dynamic disorders on the energy distribution of charge‐transfer (CT) states at donor–acceptor heterojunctions, such as those found in the active layers of organic solar cells. It is shown that each of these two disorder components can be partitioned into contributions related to the energetic disorder of the transport states and to the disorder associated with the hole–electron electrostatic interaction energies. The methodology is applied to evaluate the energy distributions of the CT states in representative bulk heterojunctions based on poly‐3‐hexyl‐thiophene and phenyl‐C₆₁‐butyric‐acid methyl ester. The results indicate that the torsional fluctuations of the polymer backbones are the main source of both static and dynamic disorders for the CT states as well as for the transport levels. The impact of static and dynamic disorders on radiative and nonradiative geminate recombination processes is also discussed.     

Development and Characterization of a Gene Expression Reporter System for Clostridium acetobutylicum ATCC 824

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of β-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional β-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the β-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the β-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the β-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of β-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.