Iron-binding biomolecules in the soluble hepatic fraction of the northern pike (Esox lucius): two-dimensional chromatographic separation with mass spectrometry detection.

Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-ß subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms.

The Human Milk Microbiota Produces Potential Therapeutic Biomolecules and Shapes the Intestinal Microbiota of Infants.

Human milk not only provides a perfect balance of nutrients to meet all the needs of the infant in the first months of life but also contains a variety of bacteria that play a key role in tailoring the neonatal faecal microbiome. Microbiome analysis of human milk and infant faeces from mother-breastfed infant pairs was performed by sequencing the V1-V3 region of the 16S rRNA gene using the Illumina MiSeq platform. According to the results, there is a connection in the composition of the microbiome in each mother-breastfed infant pair, supporting the hypothesis that the infant's gut is colonised with bacteria from human milk. MiSeq sequencing also revealed high biodiversity of the human milk microbiome and the infant faecal microbiome, whose composition changes during lactation and infant development, respectively. A total of 28 genetically distinct strains were selected by hierarchical cluster analysis of RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) electrophoresis profiles of 100 strains isolated from human milk and identified by 16S RNA sequencing. Since certain cellular molecules may support their use as probiotics, the next focus was to detect (S)-layer proteins, bacteriocins and exopolysaccharides (EPSs) that have potential as therapeutic biomolecules. SDS-PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis) coupled with LC-MS (liquid chromatography-mass spectrometry) analysis revealed that four Levilactobacillus brevis strains expressed S-layer proteins, which were identified for the first time in strains isolated from human milk. The potential biosynthesis of plantaricin was detected in six Lactiplantibacillus plantarum strains by PCR analysis and in vitro antibacterial studies. 1H NMR (Proton Nuclear Magnetic Resonance) analysis confirmed EPS production in only one strain, Limosilactobacillus fermentum MC1. The overall microbiome analysis suggests that human milk contributes to the establishment of the intestinal microbiota of infants. In addition, it is a promising source of novel Lactobacillus strains expressing specific functional biomolecules.

A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori.

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium's recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori - two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori. KEY POINTS: • A method for assessing H. pylori cells penetration by drug candidates is described. • Three alternative detection assays that complement each other can be used. • The method may be adapted for other bacteria as well.

Rapid Structure Determination of Bioactive 4″-Tetrahydrofurfuryl Macrozone Reaction Mixture Components by LC-SPE/Cryo NMR and MS.

LC-SPE/cryo NMR and MS methodologies have been developed and employed for a rapid structure determination of 4″-tetrahydrofurfuryl macrozone reaction mixture components. Macrozones, novel conjugates of azithromycin, and thiosemicarbazones have shown very good in vitro antibacterial activities against susceptible and some resistant bacterial strains and are promising agents for further development. The post-column multiple trapping of the chromatographically separated reaction mixture components on the SPE cartridges increased the sensitivity and together with cryogenically cooled NMR probe made it possible to identify and structurally characterize main 4″-tetrahydrofurfuryl macrozone reaction mixture compounds including those present at very low concentration level. This approach has several advantages over a classical off-line procedure, efficiency and low solvent consumption being the two most important ones. All identified components were process-related. It has been demonstrated that two different kinds of compounds with respect to structure were identified, i.e., macrolide-related and thiosemicarbazone-related ones. This methodology can serve as a platform for reliable and effective macrolides reaction components structure profiling, serving as both isolation and identification tools.

Vibrationally resolved valence and core photoionization and photoexcitation spectra of an electron-deficient trivalent boron compound: the case of catecholborane.

Compounds containing trivalent boron (TB) as the electron-deficient site(s) find numerous practical uses ranging from Lewis bases in organic synthesis to high-tech industry, with a number of novel applications anticipated. We present an experimental and theoretical study of the gas-phase valence photoionization (VUV-PES), core photoionization (XPS) and photoexcitation (NEXAFS) spectra of a representative TB compound catecholborane (CB). For modelling and assigning the spectra we used the ΔDFT and restricted single excitation space TD-DFT methods for the XPS and NEXAFS, and OVGF and EOM-CCSD for the VUV-PES. The vibrationally resolved structure was computed in the Franck-Condon (FC) and Herzberg-Teller (FCHT) approximations generally resulting in a good agreement with the observed spectral features. For the prediction of core-electron binding energies (CEBEs) several density functionals were tested. The best performance overall was furnished by ωB97X-D suggesting that including the dispersion correction is beneficial. The FCHT vibronic intensities are in clear discrepancy with the B 1s NEXAFS spectrum if the harmonic approximation is used for the B-H wagging mode both in the ground and in the first core-excited state. Instead, a much better agreement is obtained if the excited state potential is approximated to a symmetric double-well. The observed vibronic pattern could be a general fingerprint of the presence of TB centre(s), specifically, the transfer of the (core) density to the vacant boron p-orbital in the excited state.

Diverse coordination of aroylhydrazones toward iron(III) in solid state and in solution: spectrometric, spectroscopic and computational study.

The coordination properties of N'-(2-hydroxy-3-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (H2L1), N'-(2-hydroxy-4-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (H2L2) and N'-(2-hydroxy-5-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (H2L3) toward Fe(III) ions were studied by computational, spectrometric (MS) and spectroscopic methods (UV-Vis, IR and Raman spectroscopy) in solid state and in solution. Free ligands were present in keto-amine form with intramolecular H-bond. In MeOH:H2O 1:1 system, the 1:1 complexes with Fe(III) were formed, characterized by lgK ≥ 6. The coordination to the metal ion was achieved via oxygen and azomethine nitrogen since the hydrolysis of hydrazone bond was suppressed. Unlike the 1:1 stoichiometry in methanolic solution, the composition of the complexes extracted to chloroform was Fe(L)(HL). The release of three protons upon complexation was determined by independent spectrophotometric measurements. The complexes isolated from MeOH/EtOH solution have also stoichiometry 1:2. However, depending on the position of the methoxy substituent, two types of complexes were formed. In Fe(H2L1)2Cl3 and Fe(H2L3)2Cl3, hydrazones acted as neutral ligands, while in Fe(HL2)2Cl the keto-enol tautomeric interconversion and release of one proton per ligand took place. All complexes were analyzed in gas phase as well, using triple quadrupole, ion trap and H/D exchange for determination of labile hydrogens. Based on the fragmentation pathways, the structural isomers were distinguished.

Resolution of protein hydrogen/deuterium exchange by fitting amide exchange probabilities to the peptide isotopic envelopes.

RATIONALE: Mass spectra processing in protein hydrogen/deuterium (H/D) exchange has been remarkably improved by the introduction of fitting of the amide exchange probabilities to peptide isotopic envelope intensities (Kan et al., 2013), in contrast to methods in which only the peptide deuterium uptakes (centroid shifts of isotopic envelopes) are used. However, the known implementations are based on the general fitting routines that use only the objective function values. Besides, applicability of more than one fitting method makes necessary their comparative evaluation. METHODS: Two fitting methods were considered: the common least squares and the fitting of the multinomial distribution representing the number of deuterium atoms exchanged in the individual peptides. Both methods were applied either directly to the isotopic envelope data or to the deuterium distributions obtained by envelope deconvolution (i.e. de-isotoping). RESULTS: An autonomous Matlab script was prepared, based on the exact expressions for the gradient and Hessian of the objective function, with the trust-region algorithm implemented in the compact analytical form recently made available. The least-squares fitting to the envelope data produced the best results, with the greatest precision and good coverage of exact values by the confidence intervals. The deuterium distributions were sensitive to the (simulated) experimental error whose progression by envelope deconvolution caused degradation in accuracy. The multinomial distribution fitting exhibited poor performance due to inadequate representation of the experimental error and missing of the appropriate weight parameters. Some specific peptide arrangement details were discussed as potential sources of ambiguity in the fitting results. CONCLUSIONS: The method of fitting to peptide isotopic envelopes has been improved by using the exact gradient and Hessian of the objective function. The fitting should be repeated with different initial guesses in order to find not only the global minimum, but also the local minima with similar depths which may exist due to eventual ambiguity of the fitting results.

Characterisation of the electronic structure of galvinoxyl free radical by variable energy UPS, XPS and NEXAFS spectroscopy.

Insights into the electronic structure of galvinoxyl - a prototype persistent free radical species - are of interest to elucidate its attractive photophysical and magnetic properties and to pave way for a sensible design of novel applications. To this end, we study the photoionization and photoexcitation UPS, XPS and NEXAFS spectra of the gas-phase galvinoxyl in the valence and core (C 1s and O 1s) regions using synchrotron X-ray radiation. We observe significant variations of relative band intensities with photon energy for valence ionizations below 10 eV which are rationalized in terms of the properties of the corresponding valence molecular orbitals. We calculate the core electron binding energies and core-excited states by employing the spin-unrestricted ΔDFT (B3LYP, M06-2X, and ωB97xD) and time-dependent DFT (SRC2-BLYP) methods. A good correlation between the calculations and the measured C 1s and O 1s XPS and NEXAFS spectra is obtained if one assumes that the galvinoxyl sample has undergone a partial degradation (around 50%) to the saturated (closed-shell) phenolic-quinonic derivative known as galvinol. We carry out a comparative theoretical analysis of the XPS and NEXAFS spectra of galvinoxyl and galvinol by assigning the relevant absorptions and pointing out the most important relative differences. The calculations identify a band in the O 1s NEXAFS spectrum whose diminishing intensity is a most manifest indicator of the extent of the degradation. Such a feature may thus prove useful in monitoring the scavenging dynamics of galvinoxyl using the core-excitation spectroscopy.

Highly selective preparation of N-terminus Horseradish peroxidase-DNA conjugate with fully retained enzymatic activity: HRP-DNA structure - activity relation.

Within the last decade, the field of bio-nanoengineering has achieved significant advances allowing us to generate, e.g., nanoscaled molecular machineries with arbitrary shapes. To unleash the full potential of novel methods such as DNA origami technology, it is important to functionalise complex molecules and nanostructures precisely. Thus, considerable attention has been given to site-selective modifications of proteins allowing further incorporation of various functionalities. Here, we describe a method for the covalent attachment of oligonucleotides to the glycosylated horseradish peroxidase protein (HRP) with high N-terminus selectivity and significant yield while conserving the enzymatic activity. This two-step process includes a pH-controlled metal-free diazotransfer reaction using imidazole-1-sulfonyl azide hydrogen sulfate, which at pH 8.5 results in an N-terminal azide-functionalized protein, followed by the Cu-free click SPAAC reaction to dibenzocyclooctyne- (DBCO) modified oligonucleotides. The reaction conditions were optimised to achieve maximum yield and the best performance. The resulting protein-oligonucleotide conjugates (HRP-DNA) were characterised by electrophoresis and mass spectrometry (MS). Native-PAGE experiments demonstrated different migration patterns for HRP-DNA and the azido-modified protein allowing zymogram experiments. Structure-activity relationships of novel HRP-DNA conjugates were assessed using molecular dynamics simulations, characterising the molecular interactions that define the structural and dynamical properties of the obtained protein-oligonucleotide conjugates (POC).

Efficient and Sustainable Platform for Preparation of a High-Quality Immunoglobulin G as an Urgent Treatment Option During Emerging Virus Outbreaks.

During the pre-vaccine era of the COVID-19 pandemic convalescent plasma has once again emerged as a major potential therapeutic form of passive immunization that in specific cases still represents irreplaceable treatment option. There is a growing concern that variable concentration of neutralizing antibodies, present in convalescent plasma which originates from different donors, apparently affects its effectiveness. The drawback can be overcome through the downstream process of immunoglobulin fraction purification into a standardized product of improved safety and efficacy. All modern procedures are quite lengthy processes. They are also based on fractionation of large plasma quantities whose collection is not attainable during an epidemic. When outbreaks of infectious diseases are occurring more frequently, there is a great need for a more sustainable production approach that would be goal-oriented towards assuring easily and readily available immunoglobulin of therapeutic relevance. We propose a refinement strategy for the IgG preparation achieved through simplification and reduction of the processing steps. It was designed as a small but scalable process to offer an immediately available treatment option that would simultaneously be harmonized with an increased availability of convalescent plasma over the viral outbreak time-course. Concerning the ongoing pandemic status of the COVID-19, the proof of concept was demonstrated on anti-SARS-CoV-2 convalescent plasma but is likely applicable to any other type depending on the current needs. It was guided by the idea of persistent keeping of IgG molecules in the solution, so that protection of their native structure could be assured. Our manufacturing procedure provided a high-quality IgG product of above the average recovery whose composition profile was analyzed by mass spectrometry as quality control check. It was proved free from IgA and IgM as mediators of adverse transfusion reactions, as well as of any other residual impurities, since only IgG fragments were identified. The proportion of S protein-specific IgGs remained unchanged relative to the convalescent plasma. Undisturbed IgG subclass composition was accomplished as well. However, the fractionation principle affected the final product's capacity to neutralize wild-type SARS-CoV-2 infectivity, reducing it by half. Decrease in neutralization potency significantly correlated with the amount of IgM in the starting material.

Phosphoproteome Dynamics of Streptomyces rimosus during Submerged Growth and Antibiotic Production.

Streptomyces rimosus is an industrial streptomycete, best known as a producer of oxytetracycline, one of the most widely used antibiotics. Despite the significant contribution of Streptomyces species to the pharmaceutical industry, most omics analyses have only been conducted on the model organism Streptomyces coelicolor. In recent years, protein phosphorylation on serine, threonine, and tyrosine (Ser, Thr, and Tyr, respectively) has been shown to play a crucial role in the regulation of numerous cellular processes, including metabolic changes leading to antibiotic production and morphological changes. In this study, we performed a comprehensive quantitative (phospho)proteomic analysis during the growth of S. rimosus under conditions of oxytetracycline production and pellet fragmentation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis combined with phosphopeptide enrichment detected a total of 3,725 proteins, corresponding to 45.6% of the proteome and 417 phosphorylation sites from 230 phosphoproteins. Significant changes in abundance during three distinct growth phases were determined for 494 proteins and 98 phosphorylation sites. Functional analysis revealed changes in phosphorylation events of proteins involved in important cellular processes, including regulatory mechanisms, primary and secondary metabolism, cell division, and stress response. About 80% of the phosphoproteins detected during submerged growth of S. rimosus have not yet been reported in streptomycetes, and 55 phosphoproteins were not reported in any prokaryote studied so far. This enabled the creation of a unique resource that provides novel insights into the dynamics of (phospho)proteins and reveals many potential regulatory events during antibiotic production in liquid culture of an industrially important bacterium. IMPORTANCE Streptomyces rimosus is best known as a primary source of oxytetracycline (OTC). The significant global market value of OTC highlights the need for a better understanding of the regulatory mechanisms that lead to production of this antibiotic. Our study provides, for the first time, a detailed insight into the dynamics of (phospho)proteomic profiles during growth and antibiotic production in liquid culture of S. rimosus. Significant changes in protein synthesis and phosphorylation have been revealed for a number of important cellular proteins during the growth stages that coincide with OTC production and morphological changes of this industrially important bacterium. Most of these proteins have not been detected in previous studies. Therefore, our results significantly expand the insight into phosphorylation events associated with important cellular processes and antibiotic production; they also greatly increase the phosphoproteome of streptomycetes and contribute with newly discovered phosphoproteins to the database of prokaryotic phosphoproteomes. This can consequently lead to the design of novel research directions in elucidation of the complex regulatory network in Streptomyces.

Screening of Novel Antimicrobial Diastereomers of Azithromycin-Thiosemicarbazone Conjugates: A Combined LC-SPE/Cryo NMR, MS/MS and Molecular Modeling Approach.

A well-known class of antibacterials, 14- and 15-membered macrolides are widely prescribed to treat upper and lower respiratory tract infections. Azithromycin is a 15-membered macrolide antibiotic possessing a broad spectrum of antibacterial potency and favorable pharmacokinetics. Bacterial resistance to marketed antibiotics is growing rapidly and represents one of the major global hazards to human health. Today, there is a high need for discovery of new anti-infective agents to combat resistance. Recently discovered conjugates of azithromycin and thiosemicarbazones, the macrozones, represent one such class that exhibits promising activities against resistant pathogens. In this paper, we employed an approach which combined LC-SPE/cryo NMR, MS/MS and molecular modeling for rapid separation, identification and characterization of bioactive macrozones and their diastereomers. Multitrapping of the chromatographic peaks on SPE cartridges enabled sufficient sample quantities for structure elucidation and biological testing. Furthermore, two-dimensional NOESY NMR data and molecular dynamics simulations revealed stereogenic centers with inversion of chirality. Differences in biological activities among diastereomers were detected. These results should be considered in the process of designing new macrolide compounds with bioactivity. We have shown that this methodology can be used for a fast screening and identification of the macrolide reaction components, including stereoisomers, which can serve as a source of new antibacterials.

Binding of dipeptidyl peptidase III to the oxidative stress cell sensor Kelch-like ECH-associated protein 1 is a two-step process.

This work is about synergy of theory and experiment in revealing mechanism of binding of dipeptidyl peptidase III (DPP III) and Kelch-like ECH-associated protein 1 (KEAP1), the main cellular sensor of oxidative stress. The NRF2 ̶ KEAP1 signaling pathway is important for cell protection, but it is also impaired in many cancer cells where NRF2 target gene expression leads to resistance to chemotherapeutic drugs. DPP III competitively binds to KEAP1 in the conditions of oxidative stress and induces release of NRF2 and its translocation into nucleus. The binding is established mainly through the ETGE motif of DPP III and the Kelch domain of KEAP1. However, although part of a flexible loop, ETGE itself is firmly attached to the DPP III surface by strong hydrogen bonds. Using combined computational and experimental study, we found that DPP III ̶ Kelch binding is a two-step process comprising the endergonic loop detachment and exergonic DPP III ̶ Kelch interaction. Substitution of arginines, which keep the ETGE motif attached, decreases the work needed for its release and increases DPP III ̶ Kelch binding affinity. Interestingly, mutations of one of these arginine residues have been reported in cBioPortal for cancer genomics, implicating its possible involvement in cancer development. Communicated by Ramaswamy H. Sarma.

Effects of alpha-melanocortin enantiomers on acetaminophen-induced hepatotoxicity in CBA mice.

Proteins and peptides in mammals are based exclusively on L-amino acids. Recent investigations show that D-amino acids exhibit physiological effects in vivo, despite of their very small quantities. We have investigated the hepatoprotective effects of the Land D-enantiomers of alpha-melanocortin peptide (alpha-MSH). The results showed that peptide-enantiomerism is related to the protective effects of melanocortin peptides in vivo. L-alpha-MSH exhibited potent hepatoprotective effect in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice, while its D-mirror image was inefficient. Furthermore, the antibody to the L-peptide did not recognize the D-structure. The results indicate that the opposite peptide configuration may be used to modulate its function and metabolism in vivo and in vitro.

Aprotinin interacts with substrate-binding site of human dipeptidyl peptidase III.

Human dipeptidyl peptidase III (hDPP III) is a zinc-exopeptidase of the family M49 involved in final steps of intracellular protein degradation and in cytoprotective pathway Keap1-Nrf2. Biochemical and structural properties of this enzyme have been extensively investigated, but the knowledge on its contacts with other proteins is scarce. Previously, polypeptide aprotinin was shown to be a competitive inhibitor of hDPP III hydrolytic activity. In this study, aprotinin was first investigated as a potential substrate of hDPP III, but no degradation products were demonstrated by MALDI-TOF mass spectrometry. Subsequently, molecular details of the protein-protein interaction between aprotinin and hDPP III were studied by molecular modeling. Docking and long molecular dynamics (MD) simulations have shown that aprotinin interacts by its canonical binding epitope with the substrate binding cleft of hDPP III. Thereby, free N-terminus of aprotinin is distant from the active-site zinc. Enzyme-inhibitor complex is stabilized by intermolecular hydrogen bonding network, electrostatic and hydrophobic interactions which mostly involve constituent amino acid residues of the hDPP III substrate binding subsites S1, S1', S2, S2' and S3'. This is the first study that gives insight into aprotinin binding to a metallopeptidase. Communicated by Ramaswamy H. Sarma.

A comprehensive study of conditions of the biodegradation of a plastic additive 2,6-di-tert-butylphenol and proteomic changes in the degrader Pseudomonas aeruginosa san ai.

The Pseudomonas aeruginosa san ai strain was investigated for its capability to degrade the 2,6-di-tert-butylphenol (2,6-DTBP) plastic additive, a hazardous and toxic substance for aquatic life. This investigation was performed under different parameter values: 2,6-DTBP concentration, inoculum size, pH, and temperature. The GC-MS study showed that P. aeruginosa efficiently degraded 2,6-DTBP in the pH range of 5-8 at higher temperatures. Under exposure to 2,6-DTBP concentrations of 2, 10, and 100 mg L-1, the strain degraded by 100, 100, and 85%, respectively, for 7 days. Crude enzyme preparation from the biomass of P. aeruginosa san ai showed higher efficiency in 2,6-DTBP removal than that shown by whole microbial cells. Gene encoding for the enzymes involved in the degradation of aromatic compounds in P. aeruginosa san ai was identified. To complement the genomic data, a comparative proteomic study of P. aeruginosa san ai grown on 2,6-DTBP or sunflower oil was conducted by means of nanoLC-MS/MS. The presence of aromatic substances resulted in the upregulation of aromatic ring cleavage enzymes, whose activity was confirmed by enzymatic tests; therefore, it could be concluded that 2,6-DTBP might be degraded by ortho-ring cleavage. A comparative proteomics study of P. aeruginosa san ai indicated that the core molecular responses to aromatic substances can be summarized as the upregulation of proteins responsible for amino acid metabolism with emphasized glutamate metabolism and energy production with upregulated enzymes of glyoxylate bypass. P. aeruginosa san ai has a high capacity to efficiently degrade aromatic compounds, and therefore its whole cells or enzymes could be used in the treatment of contaminated areas.

Crystallographic snapshots of ligand binding to hexameric purine nucleoside phosphorylase and kinetic studies give insight into the mechanism of catalysis.

Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of purine nucleosides using phosphate instead of water as a second substrate. PNP from Escherichia coli is a homohexamer, build as a trimer of dimers, and each subunit can be in two conformations, open or closed. This conformational change is induced by the presence of phosphate substrate, and very likely a required step for the catalysis. Closing one active site strongly affects the others, by a yet unclear mechanism and order of events. Kinetic and ligand binding studies show strong negative cooperativity between subunits. Here, for the first time, we managed to monitor the sequence of nucleoside binding to individual subunits in the crystal structures of the wild-type enzyme, showing that first the closed sites, not the open ones, are occupied by the nucleoside. However, two mutations within the active site, Asp204Ala/Arg217Ala, are enough not only to significantly reduce the effectiveness of the enzyme, but also reverse the sequence of the nucleoside binding. In the mutant the open sites, neighbours in a dimer of those in the closed conformation, are occupied as first. This demonstrates how important for the effective catalysis of Escherichia coli PNP is proper subunit cooperation.

Non-fluorescent mutant of green fluorescent protein sheds light on the mechanism of chromophore formation.

The mechanism of green fluorescent protein (GFP) chromophore formation is still not clearly defined. Two mechanisms have been proposed: cyclisation-dehydration-oxidation (Mechanism A) and cyclisation-oxidation-dehydration (Mechanism B). To distinguish between these mechanisms, we generated a non-fluorescent mutant of GFP, S65T/G67A-GFP. This mutant folds to a stable, native-like structure but lacks fluorescence due to interruption of the chromophore maturation process. Mass spectrometric analysis of peptides derived from this mutant reveal that chromophore formation follows only mechanism A, but that the final oxidation reaction is suppressed. This result is unexpected within the pool of examined GFP mutants, since for the wild-type GFP, there is strong support for mechanism B.

Conservation of the conformational dynamics and ligand binding within M49 enzyme family.

The hydrogen deuterium exchange (HDX) mass spectrometry combined with molecular dynamics (MD) simulations was employed to investigate conformational dynamics and ligand binding within the M49 family (dipeptidyl peptidase III family). Six dipeptidyl peptidase III (DPP III) orthologues, human, yeast, three bacterial and one plant (moss) were studied. According to the results, all orthologues seem to be quite compact wherein DPP III from the thermophile Caldithrix abyssi seems to be the most compact. The protected regions are located within the two domains core and the overall flexibility profile consistent with semi-closed conformation as the dominant protein form in solution. Besides conservation of conformational dynamics within the M49 family, we also investigated the ligand, pentapeptide tynorphin, binding. By comparing HDX data obtained for unliganded protein with those obtained for its complex with tynorphin it was found that the ligand binding mode is conserved within the family. Tynorphin binds within inter-domain cleft, close to the lower domain ß-core and induces its stabilization in all orthologues. Docking combined with MD simulations revealed details of the protein flexibility as well as of the enzyme-ligand interactions.

Cadmium specific proteomic responses of a highly resistant Pseudomonas aeruginosa san ai.

Pseudomonas aeruginosa san ai is a promising candidate for bioremediation of cadmium pollution, as it resists a high concentration of up to 7.2 mM of cadmium. Leaving biomass of P. aeruginosa san ai exposed to cadmium has a large biosorption potential, implying its capacity to extract heavy metal from contaminated medium. In the present study, we investigated tolerance and accumulation of cadmium on protein level by shotgun proteomics approach based on liquid chromatography and tandem mass spectrometry coupled with bioinformatics to identify proteins. Size exclusion chromatography was used for protein prefractionation to preserve native forms of metalloproteins and protein complexes. Using this approach a total of 60 proteins were observed as up-regulated in cadmium-amended culture. Almost a third of the total numbers of up-regulated were metalloproteins. Particularly interesting are denitrification proteins which are over expressed but not active, suggesting their protective role in conditions of heavy metal exposure. P. aeruginosa san ai developed a complex mechanism to adapt to cadmium, based on: extracellular biosorption, bioaccumulation, the formation of biofilm, controlled siderophore production, enhanced respiration and modified protein profile. An increased abundance of proteins involved in: cell energy metabolism, including denitrification proteins; amino acid metabolism; cell motility and posttranslational modifications, primarily based on thiol-disulfide exchange, were observed. Enhanced oxygen consumption of biomass in cadmium-amended culture versus control was found. Our results signify that P. aeruginosa san ai is naturally well equipped to overcome and survive high doses of cadmium and, as such, has a great potential for application in bioremediation of cadmium polluted sites.