Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl− system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood–brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120 min, decaying at a rate of 5.9 × 10−3 min−1. NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC–MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo.
Chlorinated fatty aldehyde; Blood–brain barrier; Neuroinflammation; Myeloperoxidase; Plasmalogens
The structure determination of major allergens is a prerequisite for analyzing surface exposed areas of the allergen and for mapping conformational epitopes. These may be determined by experimental methods including crystallographic and NMR-based approaches or predicted by computational methods. In this review we summarize the existing structural information on allergens and their classification in protein fold families. The currently available allergen-antibody complexes are described and the experimentally obtained epitopes compared. Furthermore we discuss established methods for linear and conformational epitope mapping, putting special emphasis on a recently developed approach, which uses the structural similarity of proteins in combination with the experimental cross-reactivity data for epitope prediction.
Allergen structure; Protein family; X-ray; NMR; IgE epitope; Structure based epitope prediction
Protein carbamylation through cyanate is thought to have a causal role in promoting cardiovascular disease. We recently observed that the phagocyte protein myeloperoxidase (MPO) specifically induces high-density lipoprotein carbamylation, rather than chlorination, in human atherosclerotic lesions, raising the possibility that MPO-derived chlorinating species are involved in cyanate formation.
Here we show that MPO-derived chlorinating species rapidly decompose the plasma components thiocyanate and urea thereby promoting (lipo)protein carbamylation. Strikingly, the presence of physiologic concentrations of thiocyanate completely prevented MPO-induced 3-chlorotyrosine formation in HDL. Moreover, thiocyanate scavenged a 2.5-fold molar excess of hypochlorous acid, promoting HDL carbamylation, but not chlorination. Carbamylation of HDL resulted in a loss of anti-inflammatory and anti-oxidative properties. Cyanate significantly impaired (i) HDL’s ability to activate lecithin-cholesterol acyltransferase, (ii) the activity of paraoxonase, a major HDL-associated anti-inflammatory enzyme and (iii) the anti-oxidative activity of HDL.
Here we report that MPO-derived chlorinating species preferentially induce protein carbamylation - rather than chlorination - in the presence of physiologically relevant thiocyanate concentrations. Carbamylation of HDL results in the loss of its anti-inflammatory and anti-oxidative activities.
MPO-mediated decomposition of thiocyanate and/or urea might be a relevant mechanism for generating dysfunctional HDL in human disease.
•General method for the suppression of peaks along the diagonal for any homonuclear correlated spectra.•More reliable NOESY intensities due to the removal of artifacts from the huge diagonal peaks.•The overall appearance of the spectra is not changed.•Far less susceptible to magnetic field inhomogeneity in the z-direction by slice selective excitation.
Homonuclear two- and multidimensional NMR spectra are standard experiments for the structure determination of small to medium-sized molecules. In the large majority of homonuclear correlated spectra the diagonal contains the most intense peaks. Cross-peaks near the diagonal could overlap with huge tails of diagonal peaks and can therefore be easily overlooked. Here we present a general method for the suppression of peaks along the diagonal in homonuclear correlated spectra. It is based on a spatially selective excitation followed by the suppression of magnetization which has not changed the frequency during the mixing process. In addition to the auto correlation removal, these experiments are also less affected by magnetic field inhomogeneities due to the slice selective excitation, which on the other side leads to a reduced intensity compared to regular homonuclear correlated spectra.
Solution NMR; Diagonal peak suppression; Selective excitation; Spatially selective
A synthetic route to a trifluoromethyl and thiol containing glucose derivative (2,2,2-trifluoroethyl 6-thio-β-d-glucopyranoside) is presented, which is based on microwave-assisted Fischer glycosylation under increased pressure. This water-soluble, neutral thiol-compound can be used to selectively introduce a fluorine probe for 19F NMR spectroscopy on cysteines in proteins. It can be attached under mild conditions in an aqueous environment without the risk of denaturing the protein. This tag has been applied to determine the redox-state of two cysteine residues in a bacterial transcription activator. Qualitative information about the solvent accessibility can be obtained from F-19 solvent PREs.
Fluorinated glucose; NMR spectroscopy; Fischer glycosylation; Microwave irradiation; Cysteine redox state
The inherent cytotoxicity of aberrantly folded protein aggregates contributes substantially to the pathogenesis of amyloid diseases. It was recently shown that a class of evolutionary conserved proteins, called MOAG-4/SERF, profoundly alter amyloid toxicity via an autonomous but yet unexplained mode. We show that the biological function of human SERF1a originates from its atypical ability to specifically distinguish between amyloid and nonamyloid aggregation. This inherently unstructured protein directly affected the aggregation kinetics of a broad range of amyloidogenic proteins in vitro, while being inactive against nonamyloid aggregation. A representative biophysical analysis of the SERF1a:α-synuclein (aSyn) complex revealed that the amyloid-promoting activity resulted from an early and transient interaction, which was sufficient to provoke a massive increase of soluble aSyn amyloid nucleation templates. Therefore, the autonomous amyloid-modifying activity of SERF1a observed in living organisms relies on a direct and dedicated manipulation of the early stages in the amyloid aggregation pathway.
► SERF1a drives the assembly of amyloidogenic proteins ► SERF1a discriminates between amyloid and nonamyloid aggregation ► SERF1a acts through an early interaction with α-synuclein amyloid precursors ► SERF1a catalyzes the formation of transient α-synuclein “on-pathway” aggregates
The protein class called MOAG-4/SERF profoundly alters amyloid protein toxicity via an autonomous and previously unidentified pathway. Falsone and colleagues demonstrate that the amyloid-modifying ability of human SERF1a is driven by a transient interaction with early precursors in the amyloid pathway. As a consequence, SERF1a promotes amyloid aggregation of several amyloidogenic proteins, while being insensitive to unspecific protein aggregation. This identifies SERF1as a specialized amyloid factor, in support to its autonomous mode of action observed in cells.
Many naturally occurring bioactive peptides bind to biological membranes. Studying and elucidating the mode of interaction is often an essential step to understand their molecular and biological functions. To obtain the complete orientation and immersion depth of such compounds in the membrane or a membrane-mimetic system, a number of methods are available, which are separated in this review into four main classes: solution NMR, solid-state NMR, EPR and other methods. Solution NMR methods include the Nuclear Overhauser Effect (NOE) between peptide and membrane signals, residual dipolar couplings and the use of paramagnetic probes, either within the membrane-mimetic or in the solvent. The vast array of solid state NMR methods to study membrane-bound peptide orientation and localization includes the anisotropic chemical shift, PISA wheels, dipolar waves, the GALA, MAOS and REDOR methods and again the use of paramagnetic additives on relaxation rates. Paramagnetic additives, with their effect on spectral linewidths, have also been used in EPR spectroscopy. Additionally, the orientation of a peptide within a membrane can be obtained by the anisotropic hyperfine tensor of a rigidly attached nitroxide label. Besides these magnetic resonance techniques a series of other methods to probe the orientation of peptides in membranes has been developed, consisting of fluorescence-, infrared- and oriented circular dichroism spectroscopy, colorimetry, interface-sensitive X-ray and neutron scattering and Quartz crystal microbalance.
Antimicrobial peptides; immersion depth; membrane-bound peptides; NMR spectroscopy; orientation; paramagnetic relaxation enhancement; peptide hormones; toxins.
Indolic derivatives can affect fibril growth of amyloid forming proteins. The neurotransmitter serotonin (5-HT) is of particular interest, as it is an endogenous molecule with a possible link to neuropsychiatric symptoms of Parkinson disease. A key pathomolecular mechanism of Parkinson disease is the misfolding and aggregation of the intrinsically unstructured protein α-synuclein. We performed a biophysical study to investigate an influence between these two molecules. In an isolated in vitro system, 5-HT interfered with α-synuclein amyloid fiber maturation, resulting in the formation of partially structured, SDS-resistant intermediate aggregates. The C-terminal region of α-synuclein was essential for this interaction, which was driven mainly by electrostatic forces. 5-HT did not bind directly to monomeric α-synuclein molecules and we propose a model where 5-HT interacts with early intermediates of α-synuclein amyloidogenesis, which disfavors their further conversion into amyloid fibrils.
► The neurotransmitter serotonin (5-HT) suppresses amyloid fibril growth of alpha-synuclein (AS). ► 5-HT binds to intermediate aggregates of alpha-synuclein, not to monomeric AS. Consequently, 5-HT does not influence initial steps of amyloidogenesis. ► 5-HT promotes the accumulation of partially structured, SDS-resistant “on pathway” aggregates of AS. ► The C-terminal region of AS is essential for a charge dependent interaction. ► “On pathway” and “off-pathway” aggregations of AS might mechanistically overlap.
AS, α-synuclein; 5-HT, serotonin; 5,7-HT, 5,7 dihydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; ThioT, thioflavin T; TEM, transmission electron spectroscopy; DLS, dynamic light scattering; NAC-region, non-Aβ component region; Protein misfolding; Amyloid; Aggregation; Parkinson disease; Neurodegeneration; Indoleamine
The peptide hormone ghrelin, which is the natural ligand of the membrane-bound growth hormone secretagogue receptor (GHS-R), regulates overall body and cell growth, energy homeostasis, carbohydrate, protein and lipid metabolism and water electrolyte balance. It contains an O-acyl linked octanoyl group on Ser3 and is the only peptide known to contain such a modification. Using solution state NMR spectroscopy and ultrafiltration we found that human ghrelin binds to membrane-mimetic environments via its octanoyl group as well as the aromatic moiety of Phe4. Relaxation enhancements in a paramagnetic environment reveal that both the octanoyl group on Ser3 and the aromatic group on Phe4 are inserted deep into the hydrophobic core of phosphocholine assemblies while the remaining peptide is freely mobile in solution. In contrast, no binding was observed for des-octanoyl ghrelin. Thus, the octanoyl chain, together with the Phe4 aromatic group of ghrelin, functions as a membrane anchor. Our results are in parallel with the previous finding that a bulky hydrophobic group on Ser3 and Phe4 of ghrelin are necessary for its function and thus indicate that membrane-binding is essential for ghrelin function.
NMR spectroscopy; Membrane-mimetic; Paramagnetic relaxation enhancement; hGHSR-1a; Ghrelin; Octanoylation
Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and 1H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307T, Halococcus saccharolyticus DSM 5350T, Halococcus salifodinae DSM 8989T, Halococcus dombrowskii DSM 14522T, Halococcus hamelinensis JCM 12892T, Halococcus qingdaonensis JCM 13587T), Halorubrum sp. (Hrr. coriense DSM 10284T, Halorubrum chaoviator DSM 19316T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189T, Natronococcus occultus DSM 3396T) and Halobacterium noricense DSM 9758T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757T. Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ß-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-010-2611-6) contains supplementary material, which is available to authorized users.
Polyhydroxybutyrate; Haloarchaea; Halococcus; Halobacterium; Haloalkaliphile
Silver and gold, together with copper, form the transition metal group IB elements in the periodic
table and possess very different nuclear magnetic resonance (NMR)
spectroscopic properties. While there is only one gold isotope (197Au), which has
a spin of 3/2 and therefore a quadrupole moment, silver occurs in two isotopic forms (109Ag
and 109Au), both of which have a spin 1/2 and
similar NMR spectroscopic properties. The unfavorable properties of gold have prevented its NMR
spectroscopic investigation thus far. On the other hand, there are several reports of silver
NMR. However, the low sensitivity of silver, combined with its long relaxation times have rendered the
direct detection of silver possible only with concentrations greater than a few tenth molar. Reviewed here are
the general limitations of silver NMR and some techniques to partially overcome these
limitations, as well as a summary of currently available chemical shift and scalar coupling data on 109Ag.
The par toxin−antitoxin system is required for the stable inheritance of the plasmid pAD1 in its native host Enterococcus faecalis. It codes for the toxin Fst and a small antisense RNA which inhibits translation of toxin mRNA, and it is the only known antisense regulated toxin−antitoxin system in Gram-positive bacteria. This study presents the structure of the par toxin Fst, the first atomic resolution structure of a component of an antisense regulated toxin−antitoxin system. The mode of membrane binding was determined by relaxation enhancements in a paramagnetic environment and molecular dynamics simulation. Fst forms a membrane-binding α-helix in the N-terminal part and contains an intrinsically disordered region near the C-terminus. It binds in a transmembrane orientation with the C-terminus likely pointing toward the cytosol. Membrane-bound, α-helical peptides are frequently found in higher organisms as components of the innate immune system. Despite similarities to these antimicrobial peptides, Fst shows neither hemolytic nor antimicrobial activity when applied externally to a series of bacteria, fungal cells, and erythrocytes. Moreover, its charge distribution, orientation in the membrane, and structure distinguish it from antimicrobial peptides.
Bacterial conjugation is a form of type IV secretion that transports protein and DNA to recipient cells. Specific bacteriophage exploit the conjugative pili and cell envelope spanning protein machinery of these systems to invade bacterial cells. Infection by phage R17 requires F-like pili and coupling protein TraD, which gates the cytoplasmic entrance of the secretion channel. Here we investigate the role of TraD in R17 nucleoprotein uptake and find parallels to secretion mechanisms. The relaxosome of IncFII plasmid R1 is required. A ternary complex of plasmid oriT, TraD and a novel activation domain within the N-terminal 992 residues of TraI contributes a key mechanism involving relaxase-associated properties of TraI, protein interaction and the TraD ATPase. Helicase-associated activities of TraI are dispensable. These findings distinguish for the first time specific protein domains and complexes that process extracellular signals into distinct activation stages in the type IV initiation pathway. The study also provided insights into the evolutionary interplay of phage and the plasmids they exploit. Related plasmid F adapted to R17 independently of TraI. It follows that selection for phage resistance drives not only variation in TraA pilins but diversifies TraD and its binding partners in a plasmid-specific manner.
An unexpected, redox-neutral C=C bond isomerization of a γ-butyrolactone bearing an exo-methylene unit to the thermodynamically more favoured endo isomer (kcat = 0.076 s−1) catalysed by flavoproteins from the Old Yellow Enzyme family was discovered. Theoretical calculations and kinetic data support a mechanism through which the isomerization proceeds through FMN-mediated hydride addition onto exo-Cβ, followed by hydride abstraction from endo-Cβ′, which is in line with the well-established C=C bond bioreduction of OYEs. This new isomerase activity enriches the catalytic versatility of ene-reductases.
biocatalysis; C=C isomerization; flavins; isomerases; oxidoreductases
homonuclear decoupling; NMR spectroscopy; pure shift; structure elucidation; TOCSY
Relaxases are proteins responsible for the transfer of plasmid and chromosomal DNA from one bacterium to another during conjugation. They covalently react with a specific phosphodiester bond within DNA origin of transfer sequences, forming a nucleo-protein complex which is subsequently recruited for transport by a plasmid-encoded type IV secretion system. In previous work we identified the targeting translocation signals presented by the conjugative relaxase TraI of plasmid R1. Here we report the structure of TraI translocation signal TSA. In contrast to known translocation signals we show that TSA is an independent folding unit and thus forms a bona fide structural domain. This domain can be further divided into three subdomains with striking structural homology with helicase subdomains of the SF1B family. We also show that TSA is part of a larger vestigial helicase domain which has lost its helicase activity but not its single-stranded DNA binding capability. Finally, we further delineate the binding site responsible for translocation activity of TSA by targeting single residues for mutations. Overall, this study provides the first evidence that translocation signals can be part of larger structural scaffolds, overlapping with translocation-independent activities.
Characterisation of the structure and dynamics of large biomolecules and biomolecular complexes by NMR spectroscopy is hampered by increasing overlap and severe broadening of NMR signals. As a consequence, the number of available NMR spectroscopy data is often sparse and new approaches to provide complementary NMR spectroscopy data are needed. Paramagnetic relaxation enhancements (PREs) obtained from inert and soluble paramagnetic probes (solvent PREs) provide detailed quantitative information about the solvent accessibility of NMR-active nuclei. Solvent PREs can be easily measured without modification of the biomolecule; are sensitive to molecular structure and dynamics; and are therefore becoming increasingly powerful for the study of biomolecules, such as proteins, nucleic acids, ligands and their complexes in solution. In this Minireview, we give an overview of the available solvent PRE probes and discuss their applications for structural and dynamic characterisation of biomolecules and biomolecular complexes.
magnetic properties; NMR spectroscopy; proteins; solvent effects; structural biology
Reactions of Et3P adducts
of bissilylated germylenes and stannylenes with gold, silver, and
copper cyanides led to cyanogermyl or -stannyl complexes of the respective
metals. In the course of the reaction the phosphine moved to the metal,
while the cyanide migrated to the low-coordinate group 14 element.
The respective gold complexes were found to be monomeric, whereas
the silver and copper complexes exhibited a tendency to dimerize in
the solid state. Attempts to abstract the phosphine ligand with B(C6F5)3 led only to the formation of adducts
with the borane coordinating to the cyanide nitrogen atom.