NMR Structural Data Research Articles

Distinct modes of Si-H binding to Rh in complexes of a phosphine-diarylamido-silane (SiNP) pincer ligand.

Syntheses of Rh complexes of the phosphine-amido-silane SiNP ligand are reported. The reaction of the parent (SiNP)H ligand (4) with 0.5 equiv. [(COE)RhCl]2 (COE = cis-cyclooctene) in the presence of NaN(SiME3)2 resulted in the formation of (SiNP)Rh(COE) (5). Compound 5 was converted to a series of (SiNP)Rh(P(OR)3) complexes 6-10 (R = Ph, iPr, nBu, Et, or Me) by treatment with the corresponding phosphite. NMR and XRD structural data, as well as the DFT computational analysis indicate that compounds 5-10 are divided into two structural Types (A and B), differing in the nature of the interaction of the Si-H bond of the SiNP ligand with Rh.

Solution NMR and racemic crystallography provide insights into a novel structural class of cyclic plant peptides.

Head-to-tail cyclic and disulfide-rich peptides are natural products with applications in drug design. Among these are the PawS-Derived Peptides (PDPs) produced in seeds of the daisy plant family. PDP-23 is a unique member of this class in that it is twice the typical size and adopts two β-hairpins separated by a hinge region. The β-hairpins, both stabilised by a single disulfide bond, fold together into a V-shaped tertiary structure creating a hydrophobic core. In water two PDP-23 molecules merge their hydrophobic cores to form a square prism quaternary structure. Here, we synthesised PDP-23 and its enantiomer comprising d-amino acids and achiral glycine, which allowed us to confirm these solution NMR structural data by racemic crystallography. Furthermore, we discovered the related PDP-24. NMR analysis showed that PDP-24 does not form a dimeric structure and it has poor water solubility, but in less polar solvents adopts near identical secondary and tertiary structure to PDP-23. The natural role of these peptides in plants remains enigmatic, as we did not observe any antimicrobial or insecticidal activity. However, the plasticity of these larger PDPs and their ability to change structure under different conditions make them appealing peptide drug scaffolds.

Assessment of NR4A Ligands That Directly Bind and Modulate the Orphan Nuclear Receptor Nurr1.

Nurr1/NR4A2 is an orphan nuclear receptor transcription factor implicated as a drug target for neurological disorders including Alzheimer's and Parkinson's diseases. Previous studies identified small-molecule NR4A nuclear receptor modulators, but it remains unclear if these ligands affect transcription via direct binding to Nurr1. We assessed 12 ligands reported to affect NR4A activity for Nurr1-dependent and Nurr1-independent transcriptional effects and the ability to bind the Nurr1 ligand-binding domain (LBD). Protein NMR structural footprinting data show that amodiaquine, chloroquine, and cytosporone B bind the Nurr1 LBD; ligands that do not bind include C-DIM12, celastrol, camptothecin, IP7e, isoalantolactone, ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate (TMPA), and three high-throughput screening hit derivatives. Importantly, ligands that modulate Nurr1 transcription also show Nurr1-independent effects on transcription in a cell type-specific manner, indicating that care should be taken when interpreting the functional response of these ligands in transcriptional assays. These findings should help focus medicinal chemistry efforts that desire to optimize Nurr1-binding ligands.

Stable Lewis Base Adducts of Tetrahalodiboranes: Synthetic Methods and Structural Diversity.

A series of 22 new bis(phosphine), bis(carbene), and bis(isonitrile) tetrahalodiborane adducts has been synthesized, either by direct adduct formation with highly sensitive B2 X4 precursors (X=Cl, Br, I) or by ligand exchange at stable B2 X4 (SMe2 )2 precursors (X=Cl, Br) with labile dimethylsulfide ligands. The isolated compounds have been fully characterized using NMR spectroscopy, elemental analysis, and, for 20 of these compounds, single-crystal X-ray diffraction, revealing an unexpected variation in the bonding motifs. In addition to the classical B2 X4 L2 diborane(4) bis-adducts, certain more sterically demanding carbene ligands induce a halide displacement which led to the first halide-bridged monocationic diboron species, [B2 X3 L2 ]A (A=BCl4 , Br, I). Furthermore, low-temperature 1:1 reactions of B2 Cl4 with sterically demanding N-heterocyclic carbenes led to the formation of kinetically unstable mono-adducts, one of which was structurally characterized. A comparison of the NMR spectra and structural data of new and literature-known bis-adducts shows several trends pertaining to the nature of the halides and the stereoelectronic properties of the Lewis bases employed.

Structural Factors for Regulation of Electron Transfer from Cytochrome C to Cytochrome C Oxidase

The electron transfer (ET) reaction from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is the terminal ET reaction in the respiratory chain of mitochondria. By accepting four electrons from the repetitive binding of Cyt c, CcO reduces molecular oxygen to water molecules to promote the proton pumping from the matrix to inner membrane space in mitochondria, resulting in the formation of the proton gradient, which is the primary driving force for the ATP synthesis in the complex V. The ET reaction from Cyt c to CcO is, therefore, the crucial step for the energy generation system in the cellular level. Because the incomplete reduction of molecular oxygen generates reactive oxygen species, which is highly cytotoxic to damage cellular components, the ET reaction from Cyt c to CcO is thought to be strictly regulated and the formation of the specific ET pathway from Cyt c to CcO in the ET complex has been suggested. However, the ET pathway and its regulation mechanism are still controversial due to the lack of the structural information about the ET complex between Cyt c and CcO. To determine the structure of the Cyt c – CcO ET complex, we used the protein-protein docking simulation1 between Cyt c and CcO based on the NMR structural data for the interaction site for CcO on Cyt c 2. The predicted complex structure between Cyt c and CcO clearly showed that the interaction site between the two proteins is highly hydrophobic and the mutational study also revealed that the introduction of hydrophilic amino acid residues significantly reduced the ET rate from Cyt c to CcO3. The formation of such a hydrophobic interaction site was confirmed by the osmotic pressure experiments for the steady-state kinetic measurements of the ET reaction from Cyt c to CcO. Based on the osmotic pressure dependence of Michaelis constant, K m, for the ET reaction from Cyt c to CcO, we found out the significant dehydration from hydrophobic amino acid residues near the exposed hydrophobic heme periphery, facing to the hydrophobic amino acid residues of CcO, to form the hydrophobic interface between Cyt c and CcO in the predicted ET complex. The protein-protein docking simulation for the complex formation with a mutant having the hydrophilic amino acid residues in the interaction site for CcO indicated that a new hydrogen bond was formed in the interaction site between Cyt c and CcO, implying that the incomplete dehydration in the interaction site and/or invasion of the bulk water to the interaction site of the mutant. To determine the ET pathway from Cyt c to CcO, the conventional ET pathway analysis4 was applied to the predicted ET complex between Cyt c and CcO. Although the estimated ET pathway was constructed by many hydrophobic amino acid residues, the introduction of the hydrophobic amino acid residue into the interaction site resulted in the accelerated ET rate in the pathway analysis, which is inconsistent with the experimental data. We re-evaluate the ET pathway using the Green’s function and found that electron is directly transferred from the heme iron of Cyt c to the cupper ion of the subunit II of CcO through the tunneling mechanism, and the decreased hydrophobicity in the interaction site reduced the ET rate in this analysis. We, therefore, conclude that the formation of the hydrophobic interaction site by the dehydration around exposed heme periphery is essential for the regulation of the ET reaction from Cyt c to CcO in the respiratory chain. Sato, W.; Hitaoka, S.; Inoue, K.; Imai, M.; Saio, T.; Uchida, T.; Shinzawa-Itoh, K.; Yoshikawa, S.; Yoshizawa, K.; Ishimori, K., Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation. J. Biol. Chem. 2016, 291 (29), 15320-15331. Sakamoto, K.; Kamiya, M.; Imai, M.; Shinzawa-Itoh, K.; Uchida, T.; Kawano, K.; Yoshikawa, S.; Ishimori, K., NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 2011, 108 (30), 12271-12276. Sato, W.; Uchida, T.; Saio, T.; Ishimori, K., Polyethylene glycol promotes autoxidation of cytochrome c. Biochim. Biophys. Acta 2018, 1862 (6), 1339-1349. Onuchic, J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B., Pathway Analysis of Protein Electron-Transfer Reactions. Ann. Rev. Biophys. Biomol. Struct. 1992, 21 (1), 349-377.

Structural Characterization of Membrane-Associated BCL-2 Family Proteins

The BCL-2 family proteins are central regulators of programmed cell death. Their functions are regulated by their interactions with a wide range of proteins, both within and outside the BCL-2 family as well as with intracellular membranes, most notably, the mitochondrial outer membrane, where cell fate is ultimately decided. The family members share sequence similarity across four BCL-2 homology (BH1-BH4) motifs, of which BH3 is highly conserved and essential for cell death. Several also contain a hydrophobic C-terminus that anchors the proteins to intracellular membranes. The family includes both death antagonists that protect the cell against apoptotic stimuli, death agonists that kill the cell. Interactions with membranes and lipids are critical for BCL-2 protein function, but structural studies have focused primarily on forms of the proteins made soluble by deleting the C-terminal tail or adding detergent. As a result, the molecular mechanisms controlling BCL-2 function are incompletely understood. Here, we present NMR structural data for the anti-apoptotic protein BCL-XL, and the pro-apoptotic protein BID, associated with detergent-free lipid particles. We show that BCL-XL can be incorporated in lipid bilayer nanodiscs. BCL-XL is anchored to the nanodisc lipid bilayer membrane by tight association of its C-terminal tail, while the N-terminal head retains the canonical BCL-2 protein fold, with the surface groove solvent-exposed and available for BH3 ligand binding. The transmembrane C-terminus of BCL-XL forms an α-helix with a significant degree of conformational dynamics. We further show that tBID, the truncated active form of pro-apoptotic BID, can form soluble lipoparticles that are fully capable of engaging the canonical groove of its partner BCL-XL. The results indicate that the BCL-2 family proteins exist as lipid-associated entities, in both their cytosolic and membrane-bound states, and help reconcile a number of recent results involving membrane-associated processes.

Computational Model and Dynamics of Monomeric Full-Length APOBEC3G.

APOBEC3G (A3G) is a restriction factor that provides innate immunity against HIV-1 in the absence of viral infectivity factor (Vif) protein. However, structural information about A3G, which can aid in unraveling the mechanisms that govern its interactions and define its antiviral activity, remains unknown. Here, we built a computer model of a full-length A3G using docking approaches and molecular dynamics simulations, based on the available X-ray and NMR structural data for the two protein domains. The model revealed a large-scale dynamics of the A3G monomer, as the two A3G domains can assume compact forms or extended dumbbell type forms with domains visibly separated from each other. To validate the A3G model, we performed time-lapse high-speed atomic force microscopy (HS-AFM) experiments enabling us to get images of a fully hydrated A3G and to directly visualize its dynamics. HS-AFM confirmed that A3G exists in two forms, a globular form (∼84% of the time) and a dumbbell form (∼16% of the time), and can dynamically switch from one form to the other. The obtained HS-AFM results are in line with the computer modeling, which demonstrates a similar distribution between two forms. Furthermore, our simulations capture the complete process of A3G switching from the DNA-bound state to the closed state. The revealed dynamic nature of monomeric A3G could aid in target recognition including scanning for cytosine locations along the DNA strand and in interactions with viral RNA during packaging into HIV-1 particles.

3DIANA: 3D Domain Interaction Analysis: A Toolbox for Quaternary Structure Modeling

Electron microscopy (EM) is experiencing a revolution with the advent of a new generation of Direct Electron Detectors, enabling a broad range of large and flexible structures to be resolved well below 1 nm resolution. Although EM techniques are evolving to the point of directly obtaining structural data at near-atomic resolution, for many molecules the attainable resolution might not be enough to propose high-resolution structural models. However, accessing information on atomic coordinates is a necessary step toward a deeper understanding of the molecular mechanisms that allow proteins to perform specific tasks. For that reason, methods for the integration of EM three-dimensional maps with x-ray and NMR structural data are being developed, a modeling task that is normally referred to as fitting, resulting in the so called hybrid models. In this work, we present a novel application—3DIANA—specially targeted to those cases in which the EM map resolution is medium or low and additional experimental structural information is scarce or even lacking. In this way, 3DIANA statistically evaluates proposed/potential contacts between protein domains, presents a complete catalog of both structurally resolved and predicted interacting regions involving these domains and, finally, suggests structural templates to model the interaction between them. The evaluation of the proposed interactions is computed with DIMERO, a new method that scores physical binding sites based on the topology of protein interaction networks, which has recently shown the capability to increase by 200% the number of domain-domain interactions predicted in interactomes as compared to previous approaches. The new application displays the information at a sequence and structural level and is accessible through a web browser or as a Chimera plugin at http://3diana.cnb.csic.es.

M‐Metallaphenol: Synthesis and Reactivity Studies

Treatment of the osmium complex [Os{CHC-(PPh3 )CH(OH)-η(2) -C≡CH}(PPh3 )2 (NCS)2 ] (1) with excess triethylamine produces the first m-metallaphenol complex [Os{CHC(PPh3 )CHC(OH)CH}(PPh3 )2 (NCS)2 ] (2). The NMR spectroscopic and structural data as well as the nucleus-independent chemical-shift (NICS) values suggest that osmaphenol 2 has aromatic character. The reactivity studies demonstrate that 2 can react with different isocyanates to form the annulation reaction products [Os{CHC(PPh3 )CHC(OCONR)C}(PPh3 )2 (NCS)2 ] (R=Ph (3), iPr (7), Bn (8)) via the carbamate intermediates [Os{CHC(PPh3 )CHC(O-CONHR)CH}(PPh3 )2 (NCS)2 ] (R=Ph (4), iPr (5), Bn (6)). In addition, the similar annulation reactions can be extended to other unsaturated compounds containing N-C multiple bonds, for example, isothiocyanates, pyridine, and sodium thiocyanate, which can produce the corresponding fused osmabenzene complexes. In contrast, the reactions of 2 with common electrophiles, such as NOBF4 , NO2 BF4 , N-bromosuccinimide, and N-chlorosuccinimide only led to the decomposition of the metallaphenol ring. The experimental results suggest that 2 is very electrophilic and readily reacts with nucleophiles, which is mainly due to the metal center and the strong electron-withdrawing phosphonium group.

Familial Alzheimer’s mutations within APPTM increase Aβ42 production by enhancing accessibility of ε-cleavage site

The high Aβ42/Aβ40 production ratio is a hallmark of familial Alzheimer's disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aβ is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ε-cleavage at either T48 or L49, resulting in subsequent production of Aβ42 or Aβ40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ε-cleavage, and consequently shift cleavage preference towards Aβ42.

Cross-strand histidine-aromatic interactions enhance acyl-transfer rates in beta-hairpin peptide catalysts.

A reactive tagging methodology was used to select the species most reactive to an acylation reagent from a solid phase library of beta hairpin peptides. Hits bearing an electron-rich aromatic residue across strand from a reactive histidine were found to competitively become N-acylated. In addition to displaying rapid N-acylation rates the hit peptide was additionally deacylated in the presence of a nucleophile, thus closing a putative catalytic cycle. Variants of the hit peptide were studied to elucidate both the magnitude (up to 18,000-fold over background, kcat/kuncat = 94,000,000, or 45-fold over Boc-histidine methyl ester) and mechanism of acyl transfer catalysis. A combination of CH-π, cation-π and HisH(+)-O interactions in the cationic imidazole transition state is implicated in the rate acceleration, in addition to the fidelity of the beta hairpin fold. Moreover, NMR structural data on key intermediates or models thereof suggest that a key feature of this catalyst is the ability to access several different stabilizing conformations along the catalysis reaction coordinate.

In silico molecular docking and in vitro antidiabetic studies of dihydropyrimido[4,5-a]acridin-2-amines.

An in vitro antidiabetic activity on α-amylase and α–glucosidase activity of novel 10-chloro-4-(2-chlorophenyl)-12-phenyl-5,6-dihydropyrimido[4,5-a]acridin-2-amines (3a–3f) were evaluated. Structures of the synthesized molecules were studied by FT-IR, 1H NMR, 13C NMR, EI-MS, and single crystal X-ray structural analysis data. An in silico molecular docking was performed on synthesized molecules (3a–3f). Overall studies indicate that compound 3e is a promising compound leading to the development of selective inhibition of α-amylase and α-glucosidase.

Dynamics of Neurotoxin II Provides Adaptivity of its Functional Sites

The details of protein internal motions could be of crucial importance to understanding its function. Such a details can't be directly obtained by conventional NMR methods: the set of NMR structures and relaxation order parameters do not provide motional models. One of the viable approaches is the analysis of molecular dynamics trajectories verified by NMR structural and relaxation data sets. The technique was implemented to the peripheral protein Neurotoxin II (NTII). High-quality experimental structures of 13С/15N labeled NTII were used as starting points for a set of MD trajectories covering a total of 7 microseconds. 3JHNHA, 3JHAHB and 3JHBHG coupling constants together with backbone and sidechain 15N-/13C-relaxation rates, heteronuclear NOEs and chemical shifts were back-calculated from MD trajectories and found to be in a good agreement with experimental data. The analysis of MD trajectories revealed a number of highly flexible protein regions with locally correlated motions in the nanosecond time scale. Two of the regions, as was shown previously, provide NTII binding to the environment of the Nicotinic Acetylcholine Receptor (nAChR) and to the receptor itself. Flexibility of protein binding sites could lead to their adaptivity favoring binding to target molecules. In the case of NTII two flexible binding sites of different targeting (membrane binding site and nAChR inhibition site) act successively. High flexibility of the binding site of NTII provides its adaptivity to the specific interaction with the lipid head groups of phosphatidylserine in the environment of nAChR enabling the membrane catalysis mechanism for NTII/nAChR interaction. Then high flexibility and, accordingly, adaptivity of the nAChR inhibition site of NTII plays its role in the final step of the membrane catalysis - high affinity binding of NTII to nAChR.

Structural and Functional Studies of the Outer Membrane Protein Ail from Yersinia Pestis

Ail (Attachment invasion locus) is a virulence factor outer membrane protein of Yersinia pestis, a devastating human pathogen that causes plague. Ail plays key roles in mediating adhesion and providing resistance to human complement, making it a prime candidate target for drug development. It interacts with several human host proteins, including fibronectin, to regulate adhesion and innate immunity. Ail belongs to the Ail/Lom family (pfam PF06316) of outer membrane proteins, whose members share amino acid sequence homology in the membrane-spanning segments, but vary widely in the sequences of the extracellular loops. E. coli OmpX is regarded as the prototypical member of this family, with transmembrane eight-stranded β-barrel and four extracellular loops. However, while Ail has marked adhesion/invasion activity and is essential for virulence, OmpX has no identified function and is not essential. The four extracellular loops of Ail are thought to be responsible for function. Here we present NMR structural data obtained for Ail in detergent micelles, phospholipid vesicles and phospholipid nanodiscs. Since Ail is an integral membrane protein, understanding the molecular mechanism underpinning its function requires structure determination within the phospholipid bilayer membrane. NMR spectroscopy is unique in its ability to provide high-resolution information in lipid environments that closely resemble the cellular membranes. The NMR results are correlated with functional studies that characterize the interactions of Ail with its human protein partners essential for its function in cell adhesion.

Ensemble-Based Interpretations of NMR Structural Data to Describe Protein Internal Dynamics

NMR spectroscopy is the leading technique to characterize protein internal dynamics at the atomic level and on multiple time scales. However, the structural interpretation of the observables obtained by various measurements is not always straightforward and in many cases dynamics-related parameters are only used to “decorate” static structural models without offering explicit description of conformational heterogeneity. To overcome such limitations, several computational techniques have been developed to generate ensemble-based representations of protein structure and dynamics with the use of NMR-derived data. An important common aspect of the methods is that NMR observables and derived parameters are interpreted as properties of the ensemble instead of individual conformers. The resulting ensembles reflect the experimentally determined internal mobility of proteins at a given time scale and can be used to understand the role of internal motions in biological processes at atomic detail. In this review we provide an overview of the calculation methods currently available and examples of biological insights obtained by the ensemble-based models of the proteins investigated.

Synthesis and Characterization of Rhenabenzyne Complexes

Reactions of [ReH(5)(PMe(2)Ph)(3)] with alkynols HC≡CC(OH)(R)C≡CSiMe(3) (R=tBu, iPr, 1-adamantyl) in the presence of HCl give the vinylcarbyne complexes [Re{≡CCH=C(R)C≡CSiMe(3)}Cl(2)(PMe(2)Ph)(3)], which react with tBuMgCl to give [Re{≡CCH=C(R)C≡CSiMe(3)}HCl(PMe(2)Ph)(3)]. Treatment of [Re{≡CCH=C(R)C≡CSiMe(3)}HCl(PMe(2)Ph)(3)] with nBu(4)NF gives [Re{≡CCH=C(R)C≡CH}HCl(PMe(2)Ph)(3)], which first isomerizes to the bicyclic complexes [Re{CH=CH-C(R)=CCH=}Cl(PMe(2)Ph)(3)], and then to the rhenabenzynes [Re{≡CCH=C(R)CH=CH}Cl(PMe(2)Ph)(3)]. The NMR spectroscopic and structural data as well as the aromatic stabilization energy (ASE) and nucleus-independent chemical-shift (NICS) values suggest that these rhenabenzynes have aromatic character.

Crystal and NMR structures of a Trp-cage mini-protein benchmark for computational fold prediction

To provide high-resolution X-ray crystallographic structures of a peptide with the Trp-cage fold, we prepared a cyclized version of this motif. Cyclized Trp-cage is remarkably stable and afforded two crystal forms suitable for X-ray diffraction. The resulting higher resolution crystal structures validate the prior NMR models and provide explanations for experimental observations that could not be rationalized by NMR structural data, including the structural basis for the increase in fold stability associated with motif cyclization and the manner in which a polar serine side chain is accommodated in the hydrophobic interior. A hexameric oligomer of the cyclic peptide is found in both crystal forms and indicates that under appropriate conditions, this minimized system may also serve as a model for protein-protein interactions.

Structure and Dynamics of Membrane-Associated Hepatitis C Virus Protein P7 by NMR Spectroscopy

P7 is a 63-residue viroporin encoded by human hepatitis C virus (HCV). It has ion channel activity and plays essential roles in viral proliferation. The NMR results show that the protein consists of two transmembrane spanning regions connected by a cytoplasmic loop. The solution NMR structural data that has been acquired includes: hydrogen/deuterium exchange, paramagnetic relaxation enhancement, residual dipolar couplings, and bicelle ‘q-titration.’ These data demonstrate that the protein has a range of dynamic properties, and interestingly two segments in each of the membrane spanning helices display local motions, possibly related to function. Oriented Sample (OS) solid-state NMR spectra of p7 in aligned phospholipid bilayers provided the tilt angles of the helical segments, 25° and 10° to the membrane normal. Recent magic angle spinning (MAS) solid-state NMR data of p7 in liposomes are being used to determine the three-dimensional structure. It has been shown that known channel-blocking compounds inhibit the ion channel activity. This and the fact that knockout models of this protein inhibit infectivity of the virus indicate it may be an ideal target for future drugs. There are six major genotypes and over 100 relevant subtypes. Our current studies aim to investigate the structure and dynamic differences among the genotypes. In addition to studying different genotypes, specific mutations to the HCV J4 genotype were made in order to modulate the dynamics of the protein and obtain additional secondary structural information. These studies have the potential to identify the reasons why different genotypes have varying activity and drug binding affinities.

A Powerful Approach for the Selection of 2-Aminopurine Substitution Sites to Investigate RNA Folding

A precise tertiary structure must be adopted to allow the function of many RNAs in cells. Accordingly, increasing resources have been devoted to the elucidation of RNA structures and the folding of RNAs. 2-Aminopurine (2AP), a fluorescent nucleobase analogue, can be substituted in strategic positions of DNA or RNA molecules to act as site-specific probe to monitor folding and folding dynamics of nucleic acids. Recent studies further demonstrated the potential of 2AP modifications in the assessment of folding kinetics during ligand-induced secondary and tertiary RNA structure rearrangements. However, an efficient way to unambiguously identify reliable positions for 2AP sensors is as yet unavailable and would represent a major asset, especially in the absence of crystallographic or NMR structural data for a target molecule. We report evidence of a novel and direct correlation between the 2'-OH flexibility of nucleotides, observed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probing and the fluorescence response following nucleotide substitutions by 2AP. This correlation leads to a straightforward method, using SHAPE probing with benzoyl cyanide, to select appropriate nucleotide sites for 2AP substitution. This clear correlation is presented for three model RNAs of biological significance: the SAM-II, adenine (addA), and preQ(1) class II (preQ(1)cII) riboswitches.

Complete assignment of the 1H and 13C NMR spectra of antimicrobial 4-arylamino- 3-nitrocoumarin derivatives

Herein, we describe the synthesis and complete assignment of the (1)H and (13)C NMR chemical shifts of a series of antimicrobial 4-arylamino-3-nitrocoumarin derivatives based on a combination of (1)H and (13)C NMR, (1)H-(1)H-COSY, NOESY, HSQC and HMBC experiments. Conformational effects upon the chemical shifts of the coumarin moiety arising from the anisotropy of the aryl side group are briefly discussed. This study provides the first complete and fully assigned NMR data for this important group of antimicrobial compounds and bridges the gap existing in the literature with regard to NMR structural data for 4-arylamino-3-nitrocoumarins.