Hadamard Homonuclear Broadband Decoupled TOCSY NMR: Improved Efficacy in Detecting Long‐range Chemical Shift Correlations

The importance of Hadamard encoding pulses in one‐dimensional pure shift yielded by the chirp excitation version of selective total correlation spectroscopy (1D PSYCHE–TOCSY) experiments is discussed for chemical‐shift analysis of complex natural products at ultrahigh resolution. Herein, we adapted H_n Hadamard matrices to 1D PSYCHE–TOCSY and observed an overall circa square root of n‐fold enhancement in the signal‐to‐noise (S/N) ratio when compared to conventional 1D PSYCHE–TOCSY recorded by refocusing only one spin at a time. This enhancement in S/N facilitates the observation of very weak long‐range chemical‐shift correlations from Hadamard‐encoded PSYCHE–TOCSY (HE–PSYCHE–TOCSY). The proposed method will have a significant impact on structure determination of complex isolated/ synthetic natural products.     

Identification of fragmentation channels of dinucleotides using deuterium labeling

The fragmentation of the totally deuterated dinucleotide dAT⁻ in labile positions (heteroatom-bound hydrogens) was compared for different MS/MS methods: CID, IRMPD, and EID. These experiments allowed us to affirm the coexistence of several fragmentation channels. They can be classified according to the involvement of nonlabile or labile protons in the fragmentation process. Moreover, double resonance experiments were performed in IRMPD and EID. They demonstrated the existence of consecutive fragmentation processes. The probability with which each channel is taken depends on the fragmentation technique used, i. e., the energy and the time scale of the method. The fragmentation channels that involve labile protons requiring peculiar three-dimensional structures are entropically unfavorable and enthalpically favorable. They are more observed in IRMPD and EID. The involvement of labile and, therefore, exchangeable protons in the fragmentation mechanism casts doubt on the use of tandem mass spectrometry to localize incorporated deuteriums in oligonucleotides.     

含活泼质子水溶性化合物的核磁共振结构解析

利用核磁共振对在水溶剂易溶且含活泼质子的化合物的结构进行解析.实验中将待测样品直接溶解于二次去离子水中,并利用外标进行锁场.这样既保证了核磁共振实验的正常进行,同时避免了实验中氘代试剂对活泼质子的置换效应,为化合物中活泼质子的定位提供了直接的依据.为了保证外标锁场实验核磁共振谱图的质量,本工作采用了溶剂峰压制的核磁共振实验技术,获取了一组高质量的1D、2D谱图,并在此基础上顺利完成了对头孢米诺盐的结构确定.     

Heteronuclear 1D and 2D NMR Resonances Detected by Chemical Exchange Saturation Transfer to Water

A method to detect NMR spectra from heteronuclei through the modulation that they impose on a water resonance is exemplified. The approach exploits chemical exchange saturation transfers, which can magnify the signal of labile protons through their influence on a water peak. To impose a heteronuclear modulation on water, an HMQC‐type sequence was combined with the FLEX approach. 1D ¹⁵N NMR spectra of exchanging sites could thus be detected, with about tenfold amplifications over the ¹⁵N modulations afforded by conventionally detected HMQC NMR spectroscopy. Extensions of this approach enable 2D heteronuclear acquisitions on directly bonded ¹H–¹⁵N spin pairs, also with significant signal amplification. Despite the interesting limits of detection that these signal enhancements could open in NMR spectroscopy, these gains are constrained by the rates of solvent exchange of the targeted heteronuclear pairs, as well as by spectrometer instabilities affecting the intense water resonances detected in these experiments.     

Amino acid-type edited NMR experiments for methyl-methyl distance measurement in 13C-labeled proteins

New NMR experiments are presented for the measurement of methyl-methyl distances in (13)C-labeled proteins from a series of amino acid-type separated 2D or 3D NOESY spectra. Hadamard amino acid-type encoding of the proximal methyl groups provides the high spectral resolution required for unambiguous methyl-methyl NOE assignment, which is particularly important for fast global fold determination of proteins. The experiments can be applied to a wide range of protein systems, as exemplified for two small proteins, ubiquitin and MerAa, and the 30 kDa BRP-Blm complex.     

High-accuracy residual 1HN-13C and 1HN-1HN dipolar couplings in perdeuterated proteins

Truncation by the presence of many short-range residual dipolar couplings (RDCs) hinders the observation of long-range RDCs in weakly aligned biomacromolecules. Perdeuteration of proteins followed by reprotonation of labile hydrogen positions greatly alleviates this problem. Here we show that for small perdeuterated proteins, a large number (up to 10 in protein G) of long-range RDCs to 13C and 1HN can be observed from individual amide protons. The 1HN <--> 13C RDCs comprise correlations to 13Calpha, 13Cbeta, and 13C' nuclei of the same and the preceding amino acid, as well as 13C' nuclei of hydrogen-bonded amino acids. The accuracy of the coupling constants is very high and defines individual internuclear distances to within few picometers. Deviations between measured RDC values and values predicted from the 1.1 A crystal structure of protein G are mainly found in two surface-exposed loop regions. The deviations show a strong correlation to the B-factor of the crystal structure.     

RNA aptamers that bind flavin and nicotinamide redox cofactors

RNA molecules that specifically bind riboflavin (Rb) and beta-nicotinamide mononucleotide (NMN) have been isolated by in vitro selection. A simple structural motif containing intramolecular G-quartets was found to bind tightly to oxidized riboflavin (Kd = 1-5 micromolar). DNA versions of the consensus sequence also bind, but with weaker affinity. DMS protection experiments show that the quartet structure of these aptamers is stabilized by interaction with the flavin. As a measure of their redox specificity, the aptamers do not show significant differential binding between oxidized and reduced forms of a 5-deazariboflavin derivative that is a close structural analog of riboflavin. In contrast to the lack of redox specificity of the riboflavin aptamers, RNAs selected for binding to the nicotinamide portion of NAD discriminate between NAD and NADH in solution by over an order of magnitude. A mutagenized pool based on one of the NMN aptamer sequences was used to reselect for NMN binding. Comparison of the reselected sequences led to the identification of the binding region of the aptamer. A complex secondary structure containing two interacting stem-loops is proposed for the minimal NMN-binding RNA. The same mutagenized pool was used to select for increased discrimination between NMN and NMNH. From these reselected sequences, a mutation within the binding region was identified that increases specificity for NMN. These experiments show that RNA can bind these cofactors with low micromolar affinity and, in the case of nicotinamide cofactors, can discriminate between the two redox states. These cofactor binding motifs may provide a framework for generating new ribozymes that catalyze redox reactions similar to those found in basic metabolic pathways.     

Proton evolved local field solid-state nuclear magnetic resonance using Hadamard encoding: theory and application to membrane proteins

NMR anisotropic parameters such as dipolar couplings and chemical shifts are central to structure and orientation determination of aligned membrane proteins and liquid crystals. Among the separated local field experiments, the proton evolved local field (PELF) scheme is particularly suitable to measure dynamically averaged dipolar couplings and give information on local molecular motions. However, the PELF experiment requires the acquisition of several 2D datasets at different mixing times to optimize the sensitivity for the complete range of dipolar couplings of the resonances in the spectrum. Here, we propose a new PELF experiment that takes the advantage of the Hadamard encoding (HE) to obtain higher sensitivity for a broad range of dipolar couplings using a single 2D experiment. The HE scheme is obtained by selecting the spin operators with phase switching of hard pulses. This approach enables one to detect four spin operators, simultaneously, which can be processed into two 2D spectra covering a broader range of dipolar couplings. The advantages of the new approach are illustrated for a U-(15)N NAL single crystal and the U-(15)N labeled single-pass membrane protein sarcolipin reconstituted in oriented lipid bicelles. The HE-PELF scheme can be implemented in other multidimensional experiments to speed up the characterization of the structure and dynamics of oriented membrane proteins and liquid crystalline samples.     

Fluorine–proton correlation from isolated trifluoromethyl groups using unresolved J‐couplings

Fluorine‐containing compounds are rare in biological systems, so fluorine NMR spectroscopy can selectively detect and quantify fluorinated xenobiotics in crude biological extracts. The high sensitivity of fluorine NMR allows the detection of compounds containing isolated trifluoromethyl groups at nanogramme levels. However, it only provides limited structural information about trifluoromethyl‐containing compounds owing to the difficulty of interpreting fluorine chemical shifts and the low sensitivity of HOESY experiments used to correlate fluorine nuclei with protons in the same compound. This paper demonstrates that long‐range fluorine–proton J‐couplings can be used to correlate isolated trifluoromethyl groups with nearby protons with significantly higher sensitivity than HOESY. Fluorine‐observe fluorine–proton HMQC can even give correlations when the fluorine–proton J‐couplings are less than the observed fluorine resonance linewidth, so it provides a useful alternative source of structural information about fluorinated xenobiotics. Copyright © 2012 John Wiley & Sons, Ltd.     

Probe Design for the Effective Fluorescence Imaging of Intracellular RNA

Over the past two decades, the spatiotemporal analysis of fluorescently labeled single RNA species has provided a broad insight into the synthesis, localization, degradation, and transport of RNA. To elucidate the dynamic behavior of functional RNAs in living cells, researchers throughout the world have proposed numerous fluorometric strategies for intracellular RNA imaging. Because, like most other biological molecules, RNA is intrinsically nonfluorescent, the development of methods for the labeling of RNAs of interest with fluorescent molecules is essential. Several artificial tag sequences have been attached onto the 3′ end of target RNAs and used as scaffolds for interacting with their fluorescent counterparts. In this Personal Account, we focus on the methods that have been developed to show how RNAs expressed in cells can be labeled and visualized by fluorescent proteins, small molecules, or nucleic acids. Each of these methods is designed to increase the sensitivity and specificity for imaging or to decrease the background fluorescence.     

Probing side-chain dynamics in high molecular weight proteins by deuterium NMR spin relaxation: an application to an 82-kDa enzyme

New NMR experiments for the measurement of side-chain dynamics in high molecular weight ( approximately 100 kDa) proteins are presented. The experiments quantify (2)H spin relaxation rates in (13)CH(2)D or (13)CHD(2) methyl isotopomers and, for applications to large systems, offer significant gains both in sensitivity (2-3-fold) and resolution over previously published HSQC schemes. The methodology has been applied to investigate Ile dynamics in the 723-residue, single polypeptide chain enzyme, malate synthase G. Methyl-axis order parameters, S(axis), characterizing the amplitudes of motion of the methyl groups, have been derived from both (13)CH(2)D and (13)CHD(2) probes and are in excellent agreement. The distribution of order parameters is trimodal, reflecting the range of dynamics that are available to Ile residues. A reasonable correlation is noted between and inverse temperature factors from X-ray studies of the enzyme. The proposed methodology significantly extends the range of protein systems for which side-chain dynamics can be studied.     

Aminoglycoside microarrays to explore interactions of antibiotics with RNAs and proteins

RNA is an important target for drug discovery efforts. Several clinically used aminoglycoside antibiotics bind to bacterial rRNA and inhibit protein synthesis. Aminoglycosides, however, are losing efficacy due to their inherent toxicity and the increase in antibiotic resistance. Targeting of other RNAs is also becoming more attractive thanks to the discovery of new potential RNA drug targets through genome sequencing and biochemical efforts. Identification of new compounds that target RNA is therefore urgent, and we report here on the development of rapid screening methods to probe binding of low molecular weight ligands to proteins and RNAs. A series of aminoglycosides has been immobilized onto glass microscope slides, and binding to proteins and RNAs has been detected by fluorescence. Construction and analysis of the arrays is completed by standard DNA genechip technology. Binding of immobilized aminoglycosides to proteins that are models for study of aminoglycoside toxicity (DNA polymerase and phospholipase C), small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), and a large (approximately 400 nucleotide) group I ribozyme RNA is detected. The ability to screen large RNAs alleviates many complications associated with binding experiments that use isolated truncated regions from larger RNAs. These studies lay the foundation for rapid identification of small organic ligands from combinatorial libraries that exhibit strong and selective RNA binding while displaying decreased affinity to toxicity-causing proteins.     

Molecular recognition and screening using a 15N group selective STD NMR method

We present a novel saturation transfer difference (STD) experiment where group selective (GS) saturation of amide protons in (15)N labeled hosts is achieved. It is demonstrated that a train of BIRD(d) pulses that inverts only protons attached to (15)N indeed results in saturation of the amide protons, while the background proton magnetization is much less affected. The undesired effect of partial saturation of the unlabeled protons can be completely cancelled out in difference spectra by switching the (15)N carrier between the on- and the off-resonance frequencies. As a result, clean and artifact-free STD spectra are obtained without the need of time-consuming optimization of experimental parameters and acquiring control spectra in the absence of the host. The use of the (15)N-GS STD experiment is demonstrated for the case of a glycopeptide antibiotic (dimeric eremomycin)-cell-wall analogue peptide (N-Ac-D-Ala) model system where the host and guest (1)H signals overlap. The application seems feasible for ligand screening against proteins without the prerequisite of a clean on-resonance frequency or defined ligand library. The new experiment can be used as the basis for studying intermolecular interactions where the standard STD experiment is difficult to optimize.     

Long‐Lived States of Magnetically Equivalent Spins Populated by Dissolution‐DNP and Revealed by Enzymatic Reactions

Hyperpolarization by dissolution dynamic nuclear polarization (D ‐DNP) offers a way of enhancing NMR signals by up to five orders of magnitude in metabolites and other small molecules. Nevertheless, the lifetime of hyperpolarization is inexorably limited, as it decays toward thermal equilibrium with the nuclear spin‐lattice relaxation time. This lifetime can be extended by storing the hyperpolarization in the form of long‐lived states (LLS) that are immune to most dominant relaxation mechanisms. Levitt and co‐workers have shown how LLS can be prepared for a pair of inequivalent spins by D ‐DNP. Here, we demonstrate that this approach can also be applied to magnetically equivalent pairs of spins such as the two protons of fumarate, which can have very long LLS lifetimes. As in the case of para‐hydrogen, these hyperpolarized equivalent LLS (HELLS) are not magnetically active. However, a chemical reaction such as the enzymatic conversion of fumarate into malate can break the magnetic equivalence and reveal intense NMR signals.     

Towards the automatic analysis of 1H NMR spectra: Part 3. Confirmation of postulated chemical structure

A method is proposed which automatically compares experimental and predicted ¹H chemical shifts, integrals and scalar couplings and allows the structure on which the predictions are based to be confirmed or refuted. The method is comparatively insensitive to the variable presence of labile protons but is sensitive to the reliability of chemical shift prediction. Nonetheless, even with current commercially available ¹H chemical shift prediction, amongst a set of unrelated compounds the structure can be confirmed with 99% confidence. Where the compounds are closely related, the confidence decreases to approximately 60%, or 88% when labile protons are excluded. If closely related pairs are nominated explicitly, a specific criterion can be set for each pair, allowing every pair to be differentiated. Copyright © 2002 John Wiley & Sons, Ltd.     

Relative quantitation of proteins fractionated by the ProteomeLab™ PF 2D system using isobaric tags for relative and absolute quantitation (iTRAQ)

We describe an optimised protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass spectrometry to obtain relative quantitative data from peptides derived from tryptic digestions of proteins fractionated by using the 2D liquid-phase ProteomeLab™ PF 2D technique. This methodology is suitable for the quantitation of proteins from a pool of co-eluting proteins which are often difficult to identify for the purpose of candidate protein selection for biologically relevant qualitative/quantitative changes under experimental conditions or in disease states. iTRAQ quantitation also facilitates the possibility of result to result comparison using other methodologies such as UV protein quantitation via the ProteomeLab™ PF 2D technique. The optimised protocol outlined here allows relative quantitation by MALDI-TOF/TOF mass spectrometry with high sensitivity and without the need to perform 2D HPLC separation of labelled peptides. The overall outcome is the simplification in the data complexity and the ease of use of the labelling protocol. This study is dedicated to Dr. Josef Chmelik in memory of his contribution and constant inspiration.     

Sequence-specific detection of MicroRNAs by signal-amplifying ribozymes

The rational and straightforward design of hairpin ribozymes that can be sequence-specifically induced by external oligonucleotides is described. Due to intrinsic signal amplification, their sensitivity is at least an order of magnitude increased compared to standard molecular beacons. We applied this system to the detection of microRNAs, a recently discovered class of small endogenous RNA molecules that are involved in gene regulation. We show that the cognate microRNA can reliably and sensitively be detected at low concentrations in a mix of other microRNA sequences. These probes may be useful in applications that require direct detection of minute amounts of small DNAs or RNAs.