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The inherent poor sampling of fragment ions in time-of-flight mass analyzers was recently improved for data-dependent acquisition (DDA) by considering their drift times in traveling wave ion mobility spectrometry (TWIMS). Here, we extend this TWIMS-DDA approach to the data-independent acquisition (DIA) mode MS

The GAIN domain is a hallmark of adhesion G-protein coupled receptors (aGPCRs) as this extracellular domain contains an integral agonistic sequence (Stachel) for activation via binding to the 7-transmembrane helical (7TM) domain of the receptor. Many aGPCRs are autoproteolytically cleaved at the GPCR proteolysis site (GPS) site within the GAIN domain formed HXS/T sequence motif. However, other aGPCR can be activated without GPS cleavage. We determined the crystal structure of the human ADGRB2/BAI2 hormone receptor (HormR) and GPCR autoproteolysis-inducing (GAIN) domains and found that this aGPCR is resistant to autoproteolysis despite the presence of a canonical HLS sequence motif at the GPS. We used structural comparisons and molecular dynamics (MD) simulations to identify structural determinants that are important for autocleavage beyond the canonical HXS/T motif. These studies characterized a conserved glycine residue and an edge-π interaction of the histidine base of the GPS sequence with a phenylalanine residue that is highly conserved in cleavage-competent aGPCRs. The MD simulations showed that this interaction is important to position the imidazole group of the histidine for deprotonation of the serine or threonine nucleophile. Removal of this interaction reduced autoprote-olytic activity in the ADGRL1 receptor and restored cleavage competence of the ADGRB3 receptor in a R866H/L821F double mutant. Conservation analysis indicates that wild-type ADGRB2 and ADGRB3 are auto-cleavage-incompetent receptors.

The liver is one of the most sexually dimorphic organs. The hepatic metabolic pathways that are subject to sexual dimorphism include xenobiotic, amino acid and lipid metabolism. Non-alcoholic fatty liver disease and hepatocellular carcinoma are among diseases with sex-dependent prevalence, progression and outcome. Although male and female livers differ in their abilities to metabolize foreign compounds, including drugs, sex-dependent treatment and pharmacological dynamics are rarely applied in all relevant cases. Therefore, it is important to consider hepatic sexual dimorphism when developing new treatment strategies and to understand the underlying mechanisms in model systems. We isolated primary hepatocytes from male and female C57BL6/N mice and examined the sex-dependent transcriptome, proteome and extracellular metabolome parameters in the course of culturing them for 96 h. The sex-specific gene expression of the general xenobiotic pathway altered and the female-specific expression of Cyp2b13 and Cyp2b9 was significantly reduced during culture. Sex-dependent differences of several signaling pathways increased, including genes related to serotonin and melatonin degradation. Furthermore, the ratios of male and female gene expression were inversed for other pathways, such as amino acid degradation, beta-oxidation, androgen signaling and hepatic steatosis. Because the primary hepatocytes were cultivated without the influence of known regulators of sexual dimorphism, these results suggest currently unknown modulatory mechanisms of sexual dimorphism in vitro. The large sex-dependent differences in the regulation and dynamics of drug metabolism observed during cultivation can have an immense influence on the evaluation of pharmacodynamic processes when conducting initial preclinical trials to investigate potential new drugs. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03118-9.

Proline‐rich antimicrobial peptides expressed in insects are primarily active against Enterobacteriaceae. Mechanistically, they target the bacterial (70S) ribosome after partially transporter‐based cellular uptake, as revealed for Api137 and Onc112 on Escherichia coli. Following molecular modeling indicating that the Onc112 contact site is conserved among the ribosomes of high‐priority pathogens, the ribosome binding of Api137 and Onc112 was studied. The dissociation constants (K d) of Onc112 were ∼75 nmol/L for Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, 36 nmol/L for Pseudomonas aeruginosa, and 102 nmol/L for Staphylococcus aureus, thus indicating a very promising lead structure for developing broad‐spectrum antibiotics. Api137 bound weaker with K d values ranging from 155 nmol/L to 13 μmol/L. For most bacteria, the antibacterial activities were lower than predicted from the K d values, which was only partially explained by their ability to enter bacterial cells. Other factors limiting the activity expected from the ribosome binding might be off‐target binding., Resisting resistance: Proline‐rich antimicrobial peptides Api137 and especially Onc112 bind strongly to the bacterial (70S) ribosome, even to ribosomes of pathogens that appear to be resistant in standards antibiotic tests. The presented data indicate that Onc112 in particular might represent a valid lead structure to develop broad‐spectrum antibiotics relying on a novel bactericidal mechanism.

The rapid development, approval, and production of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than 1 year after the first reports of a new infectious disease was a real game changer, providing 80%–90% efficacy in preventing severe etiopathologies of the coronavirus disease 2019 (COVID-19). These vaccines induce an immune response against the SARS-CoV-2 spike (S) protein located on the surface of the virus particle. Antibodies (Abs) recognizing the S-protein can inhibit binding of the virus via the S-protein to the angiotensin-converting enzyme-2 (ACE-2) receptor expressed on different human cells, especially when these Abs bind to the interaction site, the so-called receptor-binding domain (RBD). We have expressed the RBDs of wild-type SARS-CoV-2 and five variants of concern (VOCs) to test the immune response in people before vaccination with mRNA vaccines BNT162b2 and mRNA-1273 and after up to three vaccinations using in-house ELISA and inhibition assays. The methods of both assays are provided. Both vaccines initiated similarly high IgG titers after two vaccinations against the wild-type and even two VOC-RBDs (alpha and delta) and strongly inhibited the corresponding RBD-ACE-2 binding. The IgG titers and inhibition of ACE-2 binding were lower for beta and gamma RBDs and much lower for omicron RBD. The third vaccination after 6 months strongly increased both the IgG titers and the neutralizing effect against all variants, especially for omicron, leading to 63% ± 13% neutralization potential. Importantly, neutralization linearly increased with the IgG titers.

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. Methods We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. Results The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995–0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. Conclusions We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.

In the present study, low concentrations of the very mild detergent n-dodecyl-α-d-maltoside in conjunction with sucrose gradient ultracentrifugation were used to prepare fucoxanthin chlorophyll protein (FCP) complexes of the centric diatom Thalassiosira pseudonana. Two main FCP fractions were observed in the sucrose gradients, one in the upper part and one at high sucrose concentrations in the lower part of the gradient. The first fraction was dominated by the 18 kDa FCP protein band in SDS-gels. Since this fraction also contained other protein bands, it was designated as fraction enriched in FCP-A complex. The second fraction contained mainly the 21 kDa FCP band, which is typical for the FCP-B complex. Determination of the lipid composition showed that both FCP fractions contained monogalactosyl diacylglycerol as the main lipid followed by the second galactolipid of the thylakoid membrane, namely digalactosyl diacylglycerol. The negatively charged lipids sulfoquinovosyl diacylglycerol and phosphatidyl glycerol were also present in both fractions in pronounced concentrations. With respect to the pigment composition, the fraction enriched in FCP-A contained a higher amount of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt), whereas the FCP-B fraction was characterized by a lower ratio of xanthophyll cycle pigments to the light-harvesting pigment fucoxanthin. Protein analysis by mass spectrometry revealed that in both FCP fractions the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (DDE) was present. In addition, the analysis showed an enrichment of DDE in the fraction enriched in FCP-A but only a very low amount of DDE in the FCP-B fraction. In-vitro de-epoxidation assays, employing the isolated FCP complexes, were characterized by an inefficient conversion of DD to Dt. However, in line with the heterogeneous DDE distribution, the fraction enriched in FCP-A showed a more pronounced DD de-epoxidation compared with the FCP-B.

α-Aminoxy peptides represent an interesting group of peptidomimetics with high proteolytic stability and the ability to fold into specific, predictable secondary structures. Here, we present a series of hybrid peptides consisting of α-aminoxy acids and α-amino acids with cationic and aromatic, hydrophobic side chains in an alternating manner synthesized using an efficient protocol that combines solution- and solid-phase synthesis. 2D ROESY experiments with a representative hexamer suggested the presence of a 7/8 helical conformation in solution. Biological evaluation revealed a significant impact of the peptide chain length and the N-terminal cap on the antimicrobial and anticancer properties of this series of hybrid peptides. The Fmoc-capped peptide 6e displayed the most potent antimicrobial activity against a panel of Gram-negative and Gram-positive bacterial strains (e. g. against E. Coli: MIC=8 mg/L; S. aureus: MIC=4 mg/L).

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Rα-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anti-cryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines pre-existing anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons.

Background Although our knowledge about diatom photosynthesis has made huge progress over the last years, many aspects about their photosynthetic apparatus are still enigmatic. According to published data, the spatial organization as well as the biochemical composition of diatom thylakoid membranes is significantly different from that of higher plants. Results In this study the pigment protein complexes of the diatom Thalassiosira pseudonana were isolated by anion exchange chromatography. A step gradient was used for the elution process, yielding five well-separated pigment protein fractions which were characterized in detail. The isolation of photosystem (PS) core complex fractions, which contained fucoxanthin chlorophyll proteins (FCPs), enabled the differentiation between different FCP complexes: FCP complexes which were more closely associated with the PSI and PSII core complexes and FCP complexes which built-up the peripheral antenna. Analysis by mass spectrometry showed that the FCP complexes associated with the PSI and PSII core complexes contained various Lhcf proteins, including Lhcf1, Lhcf2, Lhcf4, Lhcf5, Lhcf6, Lhcf8 and Lhcf9 proteins, while the peripheral FCP complexes were exclusively composed of Lhcf8 and Lhcf9. Lhcr proteins, namely Lhcr1, Lhcr3 and Lhcr14, were identified in fractions containing subunits of the PSI core complex. Lhcx1, Lhcx2 and Lhcx5 proteins co-eluted with PSII protein subunits. The first fraction contained an additional Lhcx protein, Lhcx6_1, and was furthermore characterized by high concentrations of photoprotective xanthophyll cycle pigments. Conclusion The results of the present study corroborate existing data, like the observation of a PSI-specific antenna complex in diatoms composed of Lhcr proteins. They complement other data, like e.g. on the protein composition of the 21 kDa FCP band or the Lhcf composition of FCPa and FCPb complexes. They also provide interesting new information, like the presence of the enzyme diadinoxanthin de-epoxidase in the Lhcx-containing PSII fraction, which might be relevant for the process of non-photochemical quenching. Finally, the high negative charge of the main FCP fraction may play a role in the organization and structure of the native diatom thylakoid membrane. Thus, the results present an important contribution to our understanding of the complex nature of the diatom antenna system.

Rodentibacter (R.) heylii is frequently detected in laboratory rodents. Repeats in toxin (RTX) toxins are considered important virulence factors of this major murine pathogen. We evaluated the virulence of a R. heylii strain negative for all known RTX toxin genes and Muribacter (M.) muris, a commensal in mice, in experimental infections of C57BL/6 and BALB/c mice. Experimental intranasal infection with 108 CFU of the pnxI-, pnxII- and pnxIII- R. heylii strain resulted in 75% and 100% mortality in C57BL/6 and BALB/c mice, respectively. In early losses, multiple internal organs were infected and purulent bronchopneumonia was the main pathology. Intranasal application of M. muris did not result in mortality or severe weight loss. Immunoproteomics led to the identification of a surface-associated and specific immunogen, which was designated as R. heylii immunogen A (RhiA) and which was exclusively recognised by sera obtained from mice infected with this R. heylii pathotype. RhiA is a 262.6 kDa large protein containing long imperfect tandem repeats and C-terminal RTX consensus sequences. Immunohistochemical analysis confirmed that this R. heylii pathotype expresses RhiA in the lower respiratory tract. In summary, this study describes a specific immunogen in a virulent R. heylii, strain which is an excellent antigen for pathotype-specific serological screenings and which might carry out RTX-related functions.

Precise health monitoring of laboratory animals is a critical factor for surveillance and accuracy of animal experiments. Rotavirus epizootic diarrhea of infant mice (EDIM) leads to infections in mice that can influence animal studies, e.g., by altering the intestinal physiology. Thus, the aim of this study was establishing a highly sensitive and specific ELISA for the serological detection of EDIM infections in rodents. First, virus proteins were separated by SDS-PAGE and immunogenic proteins were visualized by immunoblotting and identified after in-gel digestion by tandem mass spectrometry. Subsequently, the major immunogen VP6 (virus protein 6) was expressed in Escherichia coli in high yields, purified by affinity chromatography, and used to establish an indirect ELISA. The diagnostic sensitivity and specificity were both above 99 % and the selectivity better than 98.7 % for animals infected by other pathogens listed by the Federation of Laboratory Animal Science Associations. Importantly, the Strep-rVP6-His-ELISA was more sensitive than a commercial virus-based ELISA and is a time- and cost-efficient complement to EDIM-specific immune-fluorescence assays. In conclusion, the assay can improve health monitoring by reducing the risk of missed EDIM infections in animal housing facilities, thereby improving animal welfare, reliability of animal studies, and protection of precious mice breeds.

The designer proline-rich antimicrobial peptide (PrAMP) Chex1-Arg20 amide (ARV-1502) is active against Gram-negative and Gram-positive pathogens in different murine infection models when administered parenterally and possesses a wide therapeutic index. Here we studied the pharmacokinetics of ARV-1502 for the first time when administered intramuscularly or intravenously (IV) in Sprague Dawley rats and Beagle dogs. First, a specific and robust quantitation method relying on parallel reaction monitoring (PRM) using a high-resolution hybrid quadrupole-Orbitrap mass spectrometer coupled on-line to reversed-phase uHPLC was established and validated. The limit of detection was 2 ng/mL and the limit of quantitation was 4 ng/mL when spiked to pooled rat and dog plasma. When ARV-1502 was administered IV at doses of 75 and 250 μg/kg in dogs and rats, the plasma concentrations were 0.7 and 3.4 μg/mL 2 min post-administration, respectively. ARV-1502 plasma concentrations declined exponentially reaching levels between 2 and 4 ng/mL after 2 h. Intramuscular administration of 0.75 mg/kg in dogs and 2.5 mg/kg in rats resulted in a different pharmacokinetics profile. The plasma concentrations peaked at 15 min post-injection at 1 μg/mL (dogs) and 12 μg/mL (rats) and decreased exponentially within 3 h to 4 and 16 ng/mL, respectively. The initial plasma concentrations of ARV-1502 and the decay timing afterwards indicated that the peptide circulated in the blood stream for several hours, at some point above the minimal inhibitory concentration against multidrug-resistant Enterobacteriaceae, with blood concentrations sufficient to suppress bacterial growth and to modulate the immune system.

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with p-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. Escherichia coli was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism.

Prolinreiche antimikrobielle Peptide (PrAMPs) wurden bereits von mehreren Forschungsgruppen und Firmen als vielversprechende Leitverbindungen zur Behandlung systemischer Infektionen mit Gram-negativen Bakterien untersucht und optimiert. PrAMPs, wie Apidaecine und Oncocine, dringen in Bakterien ein und toten diese wahrscheinlich durch Inhibition spezifischer Zielstrukturen, ohne lytische Wirkung auf die Membranen. Wir zeigen hier, dass Apidaecine und Oncocine mit Dissoziationskonstanten im nanomolaren Bereich an das 70S-Ribosom binden. Apidaecine wechselwirken mindestens uber die beiden C-terminalen Reste (Arg-17 und Leu-18) stark mit dem 70S-Ribosom, wahrend die Reste Lys-3, Tyr-6, Leu-7 und Arg-11 die die wichtigsten Bindungsstellen der Oncocine sind. Letztere inhibierten die Proteinbiosynthese in vitro sehr effizient mit IC50-Werten von 150–240 nmol L−1. Das Chaperon DnaK ist hochstwahrscheinlich kein Ziel der PrAMPs, bindet diese aber mit niedrigerer Affinitat.

Bacteria have acquired resistance mechanisms to overcome antibiotic treatments, triggering major concerns about the return of epidemic infections. Antimicrobial peptides identified in insects, animals, and plants represent a huge pool of promising lead structures that can be further developed for medical applications. Short proline-rich antimicrobial peptides (PrAMPs) have gained much attention due to their clinically interesting activity spectrum, serum protease stability, efficacy in murine infection models, and low adverse effects. Here we induced resistances by incubating Escherichia coli with increasing concentrations of apidaecin 1b, a PrAMP isolated from honeybees, and quantitatively evaluated the proteomes between wild-type and resistant strains. Surprisingly, 2D differential gel electrophoresis did not reveal differences, indicating that the expression levels of dominant proteins were very similar. Reversed-phase chromatography coupled online to a mass spectrometer identified 2131 proteins in the soluble fraction (cytosolic fraction) and 1296 proteins in the nonsolubilized pellet (membrane fraction). Overall 29 proteins showed a statistically significant upregulation in the resistant E. coli strain, whereas 18 proteins were downregulated. Interestingly, periplasmic chaperone FimC, fimbrial protein FimA, and mannose-binding domain protein FimH, which are part of the fimbrial complex, were not detected in the resistant strain that was also unable to form biofilms. Furthermore, the expression of a few other proteins known as virulence factors was downregulated. Additionally, the expression level of isochorismatase hydrolase (YcaC) decreased in the membrane fraction of the resistant strain to 35%, and the corresponding knockout mutant of E. coli BW25113 was eight times less susceptible to apidaecin 1b and the related designer peptide Api88.

Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ring-shaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. Two complex structures of KlHxk1 with bound glucose provide a molecular model of substrate binding to the open conformation and the subsequent classical domain closure motion of yeast hexokinases. The entirety of the novel data extends the current concept of glucose signaling in yeast and complements the induced-fit model by integrating the events of N-terminal phosphorylation and dissociation of homodimeric yeast hexokinases.

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls.

Despite all remarkable progress in gel-based proteomics in recent years, there is still need to further improve quantification by decreasing the detection limits and increasing the dynamic range. These criteria are achieved best by fluorescent dyes that specifically stain the proteins either by adsorption after gel electrophoresis (in-gel staining) or covalent coupling prior to gel electrophoresis (in-solution staining). Here we report a multiplex analysis of protein samples using maleimide-activated cyanine-based (Cy3 and Cy5) and rhodamine-based dyes (Dy505, Dy535, and Dy635) to permanently label all thiol-groups of cysteine-containing proteins. The detection limits in SDS-PAGE were about 10 ng per band and even 2 ng for BSA due to its high content of cysteine residues. Thus only 5 microg protein of a mouse brain homogenate were analyzed by 2-DE. Both cyanine- and rhodamine-based dyes also stained proteins that did not contain cysteines, probably by reaction with amino groups. This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity. The dynamic range was more than two orders of magnitude in SDS-PAGE and the Dy-fluorophores did not alter the mobility of the tested proteins. Thus, a mixture of Dy505-, Dy555-, and Dy635-labeled Escherichia coli lysates were separated by 2-DE in a single gel and the three spot patterns relatively quantified.

Differences in the protein distribution of various protein isolates from Lupinus angustifolius L. Vitabor were identified as affected by the isolation procedure (alkaline and/or salt-induced extraction followed by isoelectric and/or dilutive precipitation). Protein isolates extracted in alkaline solution showed higher protein yields (26.4-31.7%) compared to salt-induced extraction (19.8-30.0%) or combined alkaline and salt-induced extraction (23.3-25.6%). Chemical variations among the protein isolates especially occurred within the albumins. Protein isolates precipitated isoelectrically showed the highest contents, whereas protein isolates precipitated by dilutive showed the lowest contents of conglutin delta. Furthermore, the alkaline subunits of conglutin alpha and conglutin gamma decreased during alkaline extraction compared to salt-induced extraction. A decrease in protein-bound polar and basic amino acids was shown after protein isolation. In contrast, the amounts of nonpolar, aliphatic, aromatic, hydroxylated and sulfur-rich amino acids were higher in the lupin protein isolates compared to the lupin flakes. However, the functional side chains could not be related to the specific molecular arrangements of the protein isolates, as a similar amino acid composition was found among the protein isolates.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6–7 and 19–20, and FHR-1 binds within SCR domain 3–5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.

The study investigated the influence of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt) on the spectroscopic characteristics, structure and protein composition of isolated fucoxanthin chlorophyll protein (FCP) complexes of the pennate diatom Phaeodactylum tricornutum. 77 K fluorescence emission spectra revealed that Dt-containing FCP complexes showed a characteristic long wavelength fluorescence emission at 700 nm at a pH-value of 5 whereas DD-enriched FCPs retained the typical 680 nm fluorescence emission maximum of isolated FCPs. The 700 nm emission in Dt-containing FCPs indicates an aggregation of antenna complexes and is a typical feature of the quenching site Q1 in recent models for non-photochemical fluorescence quenching (NPQ). A comparable long-wavelength fluorescence emission was found in FCP complexes prepared with either triton X-100 or n-dodecyl β-D-maltoside as detergent. A treatment of the FCP complexes at low pH-values in the presence of a high concentration of Mg2+ ions showed that the extent of FCP aggregation which leads to the 700 nm fluorescence emission is different from the macro-aggregation of antenna complexes in higher plants. Protein analyses by mass spectrometry showed that the protein composition of the DD- and Dt-enriched FCP complexes was comparable. However, the Lhcf6 and Lhcr1 polypeptides were only found in Dt-enriched FCPs isolated with dodecyl maltoside whereas the Lhcf17 protein was only detected in DD-enriched FCPs prepared with triton. With respect to low pH-induced antenna aggregation it is important that the Lhcx1 protein was found in both DD- and Dt-enriched FCPs, albeit with only two peptides with confident scores.

The nervous-system-specific microtubule-associated Tau proteins promote microtubule stability and assembly. Tau is transiently phosphorylated at about 30 positions and additionally O- and N-glycosylated. Bovine Tau was prepared from a calf brain and purified by affinity chromatography using immobilized monoclonal antibody (mAb) BT-2, which recognizes all Tau-splicing isoforms. Tau was obtained in high purities well above 90% containing even highly phosphorylated Tau versions with isoelectric points below pH 5 without discrimination. Moreover, these highly phosphorylated versions were detected only after purification. The purification progress and the final purity were studied by two-dimensional gel electrophoresis (2DE) using both protein and phosphoprotein stains as well as immunoblots.

The molecular diversity of bovine tau obtained by three different preparation protocols was characterized by immunoblot analyses after two-dimensional gel electrophoresis. These analyses revealed that tau was heterogeneously modified, that is, up to 20 spots separated along the pH gradient, mostly independent of the preparation protocol.

The microtubule-associated protein Tau (τ) regulates the assembly and disassembly of neuronal microtubules. In Alzheimer’s disease (AD), τ becomes hyperphosphorylated and aggregates to form paired helical filaments (PHF). As the phosphorylation status of normal and biopsy-derived τ versus PHF-τ is still unclear, there is need for antibodies recognizing a distinct phosphorylation pattern without cross-reactivity. Thus, we studied seven phosphorylation-dependent antibodies directed towards phosphoserine and phosphothreonine residues in positions 212, 214, 217, 231, 396, 400, and 404 of human τ (numbering according to the longest splicing-form with 441 residues). In an immunosorbent assay only one antibody showed a significant cross-reactivity towards the unmodified sequence. All other antibodies recognized only the phosphorylated sequences at lower peptide concentrations typically applied in immunosorbent assays. However, the binding of antibodies directed towards Thr212, Thr217, and Ser400 were reduced when the nearby Ser214, Thr212 or Ser396, respectively, were simultaneously phosphorylated. The phosphate specificity was confirmed on the protein level using bovine τ in its native phosphorylation status as well as τ dephosphorylated by phosphatases. Immunoblot analyses after two-dimensional gel electrophoreses also indicated that the pAbs recognized specifically the phosphorylated τ-versions.

The synthesis of some 3-aryl-3-(trifluoromethyl)3H-diazirine and benzophenone-based photoaffinity labels is reported. The photolabile group is bound to a scaffold that also accommodates functional groups to which an indicator unit (biotin) and the bioactive ligand can be attached orthogonally. To three of the labels, moenomycin was conjugated with the aim to provide tools for the identification of the moenomycin binding site within the transglycosylase domain of the enzyme PBP 1b. Some preliminary photoaffinity-labeling experiments were carried out.

The interaction of a moenomycin derivative with the enzyme penicillin binding protein 1b (PBP 1b) has been studied by means of STD NMR. The results obtained initiated the synthesis of a number of moenomycin derivatives modified in unit A including a moenomycin-ampicillin conjugate and determination of their antibiotic activities. A protocol is described that allows studying the interaction of moenomycin analogues with PBP 1b by fluorescence correlation spectroscopy.

[This corrects the article DOI: 10.1371/journal.pone.0189514.].

Animal models of familial juvenile onset of Alzheimer's disease (AD) often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation., Summary: Insight into astrocyte and radial glia pathology in an in vitro culture system derived from the APPsw pig.

Proline-rich antimicrobial peptides (PrAMPs) have been investigated and optimized by several research groups and companies as promising lead compounds to treat systemic infections caused by Gram-negative bacteria. PrAMPs, such as apidaecins and oncocins, enter the bacteria and kill them apparently through inhibition of specific targets without a lytic effect on the membranes. Both apidaecins and oncocins were shown to bind with nanomolar dissociation constants to the 70S ribosome. In apidaecins, at least the two C-terminal residues (Arg17 and Leu18) interact strongly with the 70S ribosome, whereas residues Lys3, Tyr6, Leu7, and Arg11 are the major interaction sites in oncocins. Oncocins inhibited protein biosynthesis very efficiently in vitro with half maximal inhibitory concentrations (IC50 values) of 150 to 240 nmol L(-1). The chaperone DnaK is most likely not the main target of PrAMPs but it binds them with lower affinity.

Here, we present a fast mix-and-measure immunoassay for the specific semiquantitative detection of His-tagged proteins, for example in E. coli cell lysate. The assay is based on Förster resonance energy transfer (FRET) between a lanthanide dye-labeled low-affinity His-peptide and an acceptor-labeled anti-His-tag antibody. The targeted His-tag protein in the sample displaces the donor-labeled peptide and leads to a concentration-dependent time-resolved fluorescence signal. The assay has a total assay time of less than two minutes including sample preparation. The assay recognizes both, N- and C-terminally tagged proteins. The detection limit is comparable to those obtained in SDS-PAGE or Western Blot, which are used as standard methods for the characterization of His-tag protein expression. Additionally, we demonstrate a full compatibility of the developed assay to cell lysate, and a correlation to detectable bands in a western blot application. In conclusion, this fast, sensitive, specific and affordable mix-and-measure assay provides a timesaving and user-friendly way to quantify recombinant protein expression. It substantially reduces the workload for recombinant protein detection, especially when His-tag-protein-containing fractions in manual chromatographic purifications have to be identified.

In the present work a thorough examination of the oligomeric antenna of P. tricornutum was performed. Solubilisation of thylakoids with n-dodecyl β-d-maltoside in combination with gelfiltration and sucrose density gradient centrifugation revealed the existence of two oligomeric antenna states, the FCPo and FCP complex. Calculation of the molecular masses showed that these complexes are organized as hexamers and trimers, respectively. By MS/MS analysis a specific assignment of the FCPs to their respective genes was achieved. The peripheral antenna contained the majority of the xanthophyll cycle pigments diadinoxanthin and diatoxanthin, while an additional small pool was tightly bound to the photosystem core complexes, indicating the existence of two distinct xanthophyll cycle pigment binding proteins.

The photosynthetic antenna system of diatoms contains fucoxanthin chlorophyll a/c binding proteins (FCPs), which are membrane intrinsic proteins showing high homology to the light harvesting complexes (LHC) of higher plants. In the present study, we used a mild solubilization of P. tricornutum thylakoid membranes in combination with sucrose density gradient centrifugation or gelfiltration and obtained an oligomeric FCP complex (FCPo). The spectroscopic characteristics and pigment stoichiometrics of the FCPo complex were comparable to FCP complexes that were isolated after solubilization with higher detergent per chlorophyll ratios. The excitation energy transfer between the FCP-bound pigments was more efficient in the oligomeric FCPo complexes, indicating that these complexes may represent the native form of the diatom antenna system in the thylakoid membrane. Determination of the molecular masses of the two different FCP fractions by gelfiltration revealed that the FCP complexes consisted of trimers, whereas the FCPo complexes were either composed of six monomers or two tightly associated trimers. In contrast to vascular plants, stable functional monomers could not be isolated in P. tricornutum. Both types of FCP complexes showed two protein bands in SDS-gels with apparent molecular masses of 18 and 19 kDa, respectively. Sequence analysis by MS/MS revealed that the 19 kDa protein corresponded to the fcpC and fcpD genes, whereas the 18 kDa band contained the protein of the fcpE gene. The presence of an oligomeric antenna in diatoms is in line with the oligomeric organization of antenna complexes in different photoautotrophic groups. © 2007 American Chemical Society.

The photolytic decomposition of trifunctional carbene generating photoaffinity probes in methanolic solution was studied, a cleavage reaction with butylamine in water, the conjugation with a ligand (moenomycin), and experiments that demonstrate that the fully armed probes interact with penicillin-binding protein 1b.

The photolytic decomposition of trifunctional carbene generating photoaffinity probes in methanolic solution was studied, a cleavage reaction with butylamine in water, the conjugation with a ligand (moenomycin), and experiments that demonstrate that the fully armed probes interact with penicillin-binding protein 1b.