Characterization of Structure and Dynamics of the Guanidine-II Riboswitch from Escherichia coli by NMR Spectroscopy and Small-Angle X-ray Scattering (SAXS)

Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg²⁺ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg²⁺ and ligand binding.     

Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA–ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U‐turn and a looped‐out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U‐turn fold retained biological activity. An improvement of stacking interactions in the U‐turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U‐turn motif and the looped out base, sequence–activity relationships that could not initially be explained on the basis of the structure of the aptamer–ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.     

NMR Studies of HAR1 RNA Secondary Structures Reveal Conformational Dynamics in the Human RNA

Comparative genomics has shown that noncoding RNAs can display substantial differences between humans and chimpanzees. The human accelerated region 1 (HAR1) is a section in the human genome that exhibits the most strongly accelerated rate of nucleotide substitution in relation to the chimpanzee genome. It is associated with higher cognitive functions in human brains. The HAR1 region of the HAR1F gene is transcribed into a 118 nt noncoding RNA. We provide experimental data to validate available secondary structure models of chimpanzee and human HAR1 RNA by utilizing CD and NMR spectroscopy and applying a “divide‐and‐conquer” strategy. The mutations lead to more dynamic secondary and tertiary structure in the human HAR1 RNA, presumably as part of its function. We have also determined NMR solution structures of helix H1 as the most conserved part of the chimpanzee and human HAR1 RNAs. Helix H1 contains a GAA asymmetric internal loop, the structure of which had not been solved previously. 37 nt chimpanzee and human RNA fragments (c37 and h37 RNAs) differ in a single base pair. h37 RNA folds into a slightly more stable and rigid structure than c37 RNA. Both NMR structures show structural heterogeneity of the residues corresponding to the GAA loop.     

Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi

The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.     

A Tetrahedral Boronic Acid Diester Formed by an Unnatural Amino Acid in the Ligand Pocket of an Engineered Lipocalin

Boronic acids have long been known to form cyclic diesters with cis-diol compounds, including many carbohydrates. This phenomenon was previously exploited to create an artificial lectin by incorporating p-borono-l -phenylalanine (Bpa) into the ligand pocket of an engineered lipocalin, resulting in a so-called Borocalin. Here we describe the X-ray analysis of its covalent complex with 4-nitrocatechol as a high-affinity model ligand. As expected, the crystal structure reveals the formation of a cyclic diester between the biosynthetic boronate side chain and the two ortho-hydroxy substituents of the benzene ring. Interestingly, the boron also has a hydroxide ion associated, despite an only moderately basic pH 8.5 in the crystallization buffer. The complex is stabilized by a polar contact to the side chain of Asn134 within the ligand pocket, thus validating the functional design of the Borocalin as an artificial sugar-binding protein. Our structural analysis demonstrates how a boronate can form a thermodynamically stable diester with a vicinal diol in a tetrahedral configuration in aqueous solution near physiological pH. Moreover, our data provide a basis for the further engineering of the Borocalin with the goal of specific recognition of biologically relevant glycans.     

Structure and crystallization behavior of complex mold flux glasses in the system CaO-Na2O-Li2O-CaF2-B2O3-SiO2: A multi-nuclear NMR spectroscopic study

The structure of mold flux glasses in the system CaO-(Na,Li)₂O-SiO₂-CaF₂ with unusually high modifier contents, stabilized by the addition of ∼4 mol% B₂O₃, is studied using ⁷Li, ²³Na, ¹⁹F, ¹¹B, and ²⁹Si magic-angle-spinning (MAS), and ⁷Li{¹⁹F} and ²³Na{¹⁹F} rotational echo double-resonance (REDOR) nuclear magnetic resonance (NMR) spectroscopy. When taken together, the spectroscopic results indicate that the structure of these glasses consists primarily of dimeric [Si₂O₇]⁻⁶ units that are linked to the (Ca,Na,Li)-O coordination polyhedra, and are interspersed with chains of corner-shared BO₃ units. The F atoms in the structure are exclusively bonded to Ca atoms, forming Ca(O,F)_n coordination polyhedra. This structural scenario is shown to be consistent with the crystallization of cuspidine (3CaO·2SiO₂·CaF₂) from the parent melts on slow supercooling. The progressive addition of Li to a Na-containing base composition results in a corresponding increase in the undercooling required for the nucleation of cuspidine in the melt, which is attributed to the frustrated local structure caused by the mixing of alkali ions.     

The acylation and phosphorylation pattern of lipid A from Xanthomonas campestris strongly influence its ability to trigger the innate immune response in Arabidopsis

Lipopolysaccharides (LPSs) are major components of the cell surface of Gram-negative bacteria. LPSs comprise a hydrophilic heteropolysaccharide (formed by the core oligosaccharide and the O-specific polysaccharide) that is covalently linked to the glycolipid moiety lipid A, which anchors these macromolecules to the external membrane. LPSs are one of a group of molecules called pathogen-associated molecular patterns (PAMPs) that are indispensable for bacterial growth and viability, and act to trigger innate defense responses in eukaryotes. We have previously shown that LPS from the plant pathogen Xanthomonas campestris pv. campestris (Xcc) can elicit defense responses in the model plant Arabidopsis thaliana. Here we have extended these studies by analysis of the structure and biological activity of LPS from a nonpathogenic Xcc mutant, strain 8530. We show that this Xcc strain is defective in core completion and introduces significant modification in the lipid A region, which involves the degree of acylation and nonstoichiometric substitution of the phosphate groups with phosphoethanolamine. Lipid A that was isolated from Xcc strain 8530 did not have the ability to induce the defense-related gene PR1 in Arabidopsis, or to prevent the hypersensitive response (HR) that is caused by avirulent bacteria as the lipid A from the wild-type could. This suggests that Xcc has the capacity to modify the structure of the lipid A to reduce its activity as a PAMP. We speculate that such effects might occur in wild-type bacteria that are exposed to stresses such as those that might be encountered during plant colonization and disease.     

Structure Determination of the Top‐Loop of the Conserved 3′‐Terminal Secondary Structure in the Genome of Flaviviruses

To what extent small differences in RNA sequences (mutations) can have a profound impact on biology remains an intriguing question. This effect can be studied by using untranslated RNA regions as a model. We have studied the influence of mutations on the structure of an RNA hairpin that occurs in the 3′‐untranslated region (UTR) of Flaviviridae, and is known to have a large impact on the vector dependency of flaviviruses. Three related RNA sequences were studied by NMR spectroscopy. The selected sequences represent each one of the three clusters in the flavivirus genes (mosquito‐borne, tick‐borne, and no‐known‐vector viruses). A new strategy was used to obtain chemical shift signatures of carbonyl atoms in unlabeled uridine nucleobases to characterize their involvement in hydrogen bonding. Clear differences occur in the structures and stacking pattern of the three RNA hairpins. The observed differences cannot be predicted based on sequence analysis. A different biology can be correlated with a different RNA tertiary structure. The underlying biological mechanism, however, remains to be studied.     

A Novel Coenzyme A Analogue in the Anaerobic,Sulfate-Reducing,Marine Bacterium Desulfobacula toluolica Tol2T

Coenzyme A (CoA) thioesters are formed during anabolic and catabolic reactions in every organism. Degradation pathways of growth-supporting substrates in bacteria can be predicted by differential proteogenomic studies. Direct detection of proposed metabolites such as CoA thioesters by high-performance liquid chromatography coupled with high-resolution mass spectrometry can confirm the reaction sequence and demonstrate the activity of these degradation pathways. In the metabolomes of the anaerobic sulfate-reducing bacterium Desulfobacula toluolica Tol2^T grown with different substrates various CoA thioesters, derived from amino acid, fatty acid or alcohol metabolism, have been detected. Additionally, the cell extracts of this bacterium revealed a number of CoA analogues with molecular masses increased by 1 dalton. By comparing the chromatographic and mass spectrometric properties of synthetic reference standards with those of compounds detected in cell extracts of D. toluolica Tol2^T and by performing co-injection experiments, these analogues were identified as inosino-CoAs. These CoA thioesters contain inosine instead of adenosine as the nucleoside. To the best of our knowledge, this finding represents the first detection of naturally occurring inosino-CoA analogues.     

Quantitative Metabolomic Analysis of Changes in the Rat Blood Serum during Autophagy Modulation: A Focus on Accelerated Senescence

Autophagy is involved in the maintenance of cellular homeostasis and the removal of damaged proteins and organelles and is necessary to maintain cell metabolism in conditions of energy and nutrient deficiency. A decrease in autophagic activity plays an important role in age-related diseases. However, the metabolic response to autophagy modulation remains poorly understood. Here, we for the first time explored the effects of (1) autophagy activation by 48 h fasting, (2) inhibition by chloroquine (CQ) treatment, and (3) combined effects of fasting and CQ on the quantitative composition of metabolites in the blood serum of senescent-accelerated OXYS and control Wistar rats at the age of 4 months. By means of high-resolution ¹H NMR spectroscopy, we identified the quantitative content of 55 serum metabolites, including amino acids, organic acids, antioxidants, osmolytes, glycosides, purine, and pyrimidine derivatives. Groups of 48 h fasting (induction of autophagy), CQ treatment (inhibition of autophagy), and combined effects (CQ + fasting) are clearly separated from control groups by principal component analysis. Fasting for 48 h led to significant changes in the serum metabolomic profile, primarily affecting metabolic pathways related to fatty acid metabolism, and led to metabolism of several amino acids. Under CQ treatment, the most affected metabolites were citrate, betaine, cytidine, proline, tryptophan, glutamate, and mannose. As shown by two-way ANOVA, for many metabolites the effects of autophagy modulation depend on the animal genotype, indicating a dysregulation of metabolome reactivity in OXYS rats. Thus, the metabolic responses to modulation of autophagy in OXYS rats and Wistar rats are different. Altered metabolites in OXYS rats may serve as potential biomarkers of the manifestation of the signs of accelerated aging. Metabolic signatures characteristic to fasting and CQ treatment revealed in this work might provide a better understanding of the connections between metabolism and autophagy.     

Ten-vertex osmamonocarbaboranes via arachno and nido {CB9} monocarbaboranes. Polyhedral contraction promoted by [OsCl2(PPh3)3] in MeOH and the crystal and molecular structure of [1-H-1,1-(PPh3)2-2-Ph-3-(OMe)-isocloso-1,2-OsCB8H7]

Reactions of [OsCl₂(PPh₃)₃] 1 with the [NEt₄]⁺ salts of the [6-Ph-nido-6-CB₉H₁₁]⁻ anion 2 and the [arachno-6-CB₉H₁₄]⁻ anion 3 each proceed selectively in methanol solution with the elimination of one boron-containing fragment from the starting cluster polyhedron, yielding together a short series of three novel 10-vertex hydridoosmamonocarbaboranes of isocloso {OsCB₈} cluster geometry.     

The alpha-to-beta conformational transition of Alzheimer's Abeta-(1-42) peptide in aqueous media is reversible: a step by step conformational analysis suggests the location of beta conformation seeding

Current views of the role of beta-amyloid (Abeta) peptide fibrils range from regarding them as the cause of Alzheimer's pathology to having a protective function. In the last few years, it has also been suggested that soluble oligomers might be the most important toxic species. In all cases, the study of the conformational properties of Abeta peptides in soluble form constitutes a basic approach to the design of molecules with "antiamyloid" activity. We have experimentally investigated the conformational path that can lead the Abeta-(1-42) peptide from the native state, which is represented by an alpha helix embedded in the membrane, to the final state in the amyloid fibrils, which is characterized by beta-sheet structures. The conformational steps were monitored by using CD and NMR spectroscopy in media of varying polarities. This was achieved by changing the composition of water and hexafluoroisopropanol (HFIP). In the presence of HFIP, beta conformations can be observed in solutions that have very high water content (up to 99 % water; v/v). These can be turned back to alpha helices simply by adding the appropriate amount of HFIP. The transition of Abeta-(1-42) from alpha to beta conformations occurs when the amount of water is higher than 80 % (v/v). The NMR structure solved in HFIP/H2O with high water content showed that, on going from very apolar to polar environments, the long N-terminal helix is essentially retained, whereas the shorter C-terminal helix is lost. The complete conformational path was investigated in detail with the aid of molecular-dynamics simulations in explicit solvent, which led to the localization of residues that might seed beta conformations. The structures obtained might help to find regions that are more affected by environmental conditions in vivo. This could in turn aid the design of molecules able to inhibit fibril deposition or revert oligomerization processes.     

Influence of reaction conditions on the formation of nanotubes/nanoparticles of polyaniline in the presence of 1‐amino‐2‐naphthol‐4‐sulfonic acid and applications as electrostatic charge dissipation material

BACKGROUND: Poly(1‐amino‐2‐naphthol‐4‐sulfonic acid) and its copolymers with aniline are a new class of conducting polymers which can acquire intrinsic protonic doping ability, leading to the formation of highly soluble self‐doped homopolymers and copolymers. Free ? OH and ? NH₂ groups in the polymer chain can combine with other functional groups that could be present in protective paints which can thus be successfully used as antistatic materials. RESULTS: This paper reports the formation of nanotubes of polyaniline on carrying out oxidative polymerization of aniline in the presence of 1‐amino‐2‐naphthol‐4‐sulfonic acid (ANSA) in p‐toluenesulfonic acid (PTSA) as an external dopant. The presence of ? SO₃H groups in the ANSA comonomer allows the copolymer to acquire intrinsic protonic doping ability. The polymerization mechanism was investigated by analysing the ¹H NMR, ¹³C NMR, Fourier transform infrared and X‐ray photoelectron spectra of the copolymers and homopolymers, which revealed the involvement of ? OH/? NH₂ in the reaction mechanism. Scanning and transmission electron microscopy showed how the reaction route and the presence of a dopant can affect the morphology and size of the polymers. Static decay time measurements were also carried out on conducting copolymer films prepared by blending of 1 wt% of copolymers of ANSA and aniline with low‐density polyethylene (LDPE) which showed a static decay time of 0.1 to 0.31 s on dissipating a charge from 5000 to 500 V. CONCLUSION: Copolymers of ANSA with aniline were synthesized in different reaction media, leading to the formation of nanotubes and nanoparticles of copolymer. Blends of 1 wt% of PTSA‐ and self‐doped copolymers of ANSA and aniline with LDPE can be formulated into films with effective antistatic properties. Copyright © 2009 Society of Chemical Industry     

Structural Studies of the Lipopolysaccharide of Aeromonas veronii bv. sobria Strain K133 Which Represents New Provisional Serogroup PGO1 Prevailing among Mesophilic Aeromonads on Polish Fish Farms

In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and ¹H and ¹³C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes.     

Rational Design of CYP3A4 Inhibitors: A One-Atom Linker Elongation in Ritonavir-Like Compounds Leads to a Marked Improvement in the Binding Strength

Inhibition of the major human drug-metabolizing cytochrome P450 3A4 (CYP3A4) by pharmaceuticals and other xenobiotics could lead to toxicity, drug–drug interactions and other adverse effects, as well as pharmacoenhancement. Despite serious clinical implications, the structural basis and attributes required for the potent inhibition of CYP3A4 remain to be established. We utilized a rational inhibitor design to investigate the structure–activity relationships in the analogues of ritonavir, the most potent CYP3A4 inhibitor in clinical use. This study elucidated the optimal length of the head-group spacer using eleven (series V) analogues with the R₁/R₂ side-groups as phenyls or R₁–phenyl/R₂–indole/naphthalene in various stereo configurations. Spectral, functional and structural characterization of the inhibitory complexes showed that a one-atom head-group linker elongation, from pyridyl–ethyl to pyridyl–propyl, was beneficial and markedly improved K_s, IC₅₀ and thermostability of CYP3A4. In contrast, a two-atom linker extension led to a multi-fold decrease in the binding and inhibitory strength, possibly due to spatial and/or conformational constraints. The lead compound, 3h, was among the best inhibitors designed so far and overall, the strongest binder (K_s and IC₅₀ of 0.007 and 0.090 µM, respectively). 3h was the fourth structurally simpler inhibitor superior to ritonavir, which further demonstrates the power of our approach.