Cryptophane-xenon complexes in organic solvents observed through NMR spectroscopy

The interaction of xenon with cryptophane derivatives is analyzed by NMR by using either thermal or hyperpolarized noble gas. Twelve hosts differing by their stereochemistry, cavity size, and the nature and the number of the substituents on the aromatic rings have been included in the study, in the aim of extracting some clues for the optimization of (129)Xe-NMR based biosensors derived from these cage molecules. Four important properties have been examined: xenon-host binding constant, in-out exchange rate of the noble gas, chemical shift, and relaxation of caged xenon. This work aims at understanding the main characteristics of the host-guest interaction in order to choose the best candidate for the biosensing approach. Moreover, rationalizing xenon chemical shift as a function of structural parameters would also help for setting up multiplexing applications. Xenon exhibits the highest affinity for the smallest cryptophane, namely cryptophane-111, and a long relaxation time inside it, convenient for conservation of its hyperpolarization. However, very slow in-out xenon exchange could represent a limitation for its future applicability for the biosensing approach, because the replenishment of the cage in laser-polarized xenon, enabling a further gain in sensitivity, cannot be fully exploited.     

Determining accurate molecular sizes in solution through NMR diffusion spectroscopy

This tutorial review deals with the methodological procedures that can be used to obtain accurate molecular sizes in solution from diffusion NMR spectroscopy. The critical aspects associated with the estimation of the size of molecules from the measured translational self-diffusion coefficient, using the Stokes-Einstein equation, are highlighted and discussed. From a theoretical point of view, it is shown how to take into account the size of the solute with respect to that of the solvent and its non-spherical shape using the appropriate correction factors in the frictional coefficient. From a practical point of view, the advantages of introducing an internal standard in the sample are presented. Initially, non-aggregating systems are considered in an attempt to clarify what hydrodynamic dimensions mean. Successively, aggregating systems are addressed showing how it is possible to understand the aggregation level and derive the thermodynamic parameters for some illustrative aggregation processes.     

Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states

cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a significantly more extended allosteric network that, unlike PKA, involves a tight coupling between the alpha- and beta-subdomains of the EPAC CBD. The proposed mechanism of allosteric activation will serve as a basis to understand agonism and antagonism in the EPAC system and provides also a general paradigm for how small ligands control protein-protein interfaces.     

Hyperdimensional NMR spectroscopy

Hyperdimensional NMR describes the multiple interactions between a large number, N, of coupled nuclear spins. We show how to construct a hypothetical N-dimensional spectrum from a limited set of low-dimensional measurements, exploiting the concept of projection-reconstruction. In the process, many correlation spectra are obtained that were not investigated directly. As an illustration, the hypothetical ten-dimensional spectrum of a small protein (agitoxin) has been reconstructed, and a large number of pairwise correlation spectra have been recorded.     

Thallium NMR spectroscopy

Nuclear magnetic resonance spectroscopy has proven to be a powerful technique for solving a wide variety of problems. However, the full potential of the technique as extended to the study, through direct observation, of heavy metals in both the solid and solution state is just beginning to be realized.     

Kinetics of RNA refolding in dynamic equilibrium by 1H-detected 15N exchange NMR spectroscopy

By implementing new NMR methods that were designed to map very slow exchange processes we have investigated and characterized the refolding kinetics of a thermodynamically stable 34mer RNA sequence in dynamic equilibrium. The RNA sequence was designed to undergo a topologically favored conformational exchange between different hairpin folds, serving as a model to estimate the minimal time required for more complex RNA folding processes. Chemically prepared RNA sequences with sequence-selective (15)N labels provided the required signal separation and allowed a straightforward signal assignment of the imino protons by HNN correlation experiments. The 2D version of the new (1)H-detected (15)N exchange spectroscopy (EXSY) pulse sequence provided cross-peaks for resonances belonging to different folds that interchange on the time scale of longitudinal relaxation of (15)N nuclei bound to imino protons. The 34mer RNA sequence exhibits two folds which exchange on the observable time scale (tau(obs) approximately T(1){(15)Nu} < 5 s) and a third fold which is static on this time scale. A 1D version of the (15)N exchange experiment allowed the measurement of the exchange rates between the two exchanging folds as a function of temperature and the determination of the corresponding activation energies E(a) and frequency factors A. We found that the refolding rates are strongly affected by an entropically favorable preorientation of the replacing strand. The activation energies are comparable to values obtained for the slow refolding of RNA sequences of similar thermodynamic stability but less favorable topology.     

Protein backbone dynamics through 13C'-13Calpha cross-relaxation in NMR spectroscopy

Internal dynamics of proteins are usually characterized by the analysis of (15)N relaxation rates that reflect the motions of NH(N) vectors. It was suggested a decade ago that additional information on backbone motions can be obtained by measuring cross-relaxation rates associated with intra-residue C'C(alpha) vectors. Here we propose a new approach to such measurements, based on the observation of the transfer between two-spin orders 2N(z)() and 2N(z)(). This amounts to "anchoring" the and operators to the N(z)() term from the amide of the next residue. In combination with symmetrical reconversion, this method greatly reduces various artifacts. The experiment is carried out on human ubiquitin at 284.1 K, where the correlation time is 7.1 ns. The motions of the C'C(alpha) vector appear more restricted than those of the NH(N) vector.     

吡啶酮系偶氮染料解离平衡的^15NNMR和IR波谱

报道了四个富氮-15吡啶酮偶氮染料的氮-15核磁共振中β-氮原子的化学位移,研究了氮-15原子和pH值之间的关联.当样品溶液从酸性转成碱性,β-氮-15原子的化学位移从366-380PPM移到500PPM,相应于从腺型到偶氮阴离子结构.根据各种pH值的化学位移可计算腺结构的含量,用IR证实了这一系列化合物的酸碱离解平衡.在酸性或中性条件下,固态化合物的二个羰基吸收,在碱性条件下变成只有一个羰基吸收峰,其它官能团的吸收峰表明了与平衡的位移的相应变化.     

Indirect covariance NMR spectroscopy

A novel NMR scheme is presented that establishes homonuclear spin correlations without requiring direct detection of the spin species. This covariance NMR method is experimentally demonstrated for a mixture of amino acids and for the uniformly 13C-labeled cyclic decapeptide antamanide using a 13C-edited TOCSY experiment. The method opens up new avenues for the experimental analysis of molecules containing insensitive spins encountered in biomolecular NMR and analytical chemistry including metabolomics.     

Simultaneously cycled NMR spectroscopy

Simultaneously cycled (SC) NMR was introduced and exemplified by implementing a set of 2-D [1H,1H] SC exclusive COSY (E.COSY) NMR experiments, that is, rf pulse flip-angle cycled (SFC), rf pulse phase cycled (SPC), and pulsed field gradient (PFG) strength cycled (SGC) E.COSY. Spatially selective 1H rf pulses were applied as composite pulses such that all steps of the respective cycles were affected simultaneously in different slices of the sample. This increased the data acquisition speed for an n-step cycle n-fold. A high intrinsic sensitivity was achieved by defining the cycles in a manner that the receiver phase remains constant for all steps of the cycle. Then, the signal resulting from applying the cycle corresponded to the sum of the signals from all steps of the cycle. Hence, the detected free induction decay did not have to be separated into the contributions arising from different slices, and read-out PFGs, which not only greatly reduce sensitivity but also negatively impact lineshapes in the direct dimension, were avoided. The current implementation of SFC E.COSY reached approximately 65% of the intrinsic sensitivity of the conventional phase cycled congener, making this experiment highly attractive whenever conventional data acquisition is sampling limited. Highly resolved SC E.COSY yielding accurate 3J-coupling values was recorded for the 416 Da plant alkaloid tomatidine within 80 min, that is, 12 times faster than with conventional phase cycled E.COSY. SC NMR is applicable for a large variety of NMR experiments and thus promises to be a valuable addition to the arsenal of approaches for tackling the NMR sampling problem to avoid sampling limited data acquisition.     

Conformational equilibrium in 8-methyl-cis-2-thiahydrindane and 8-methyl-cis-2-oxahydrindane by 13C NMR spectroscopy

The preferred conformation of 8-methyl-cis-thiahydrindane has been both estimated by ¹³C NMR chemical shifts and determined by low temperature ¹³C NMR spectroscopy to be the conformer with the methyl group equatorial with respect to the cyclohexane ring. This result is in disagreement with the interpretation of the temperature dependence of the CD spectra of (+) and (?) 8-methyl-cis-2-thiahydrindane, whereby the conformation with the methyl group axial with respect to the cyclohexane ring was claimed to be the preferred conformation. The preferred conformation of the related oxygen heterocycle, 8-methyl-cis-2-oxahydrindane, has been estimated by ¹³C NMR chemical shifts to be the conformer with the methyl group axial with respect to the cyclohexane ring. Possible reasons for these observations are discussed.     

Conformational equilibrium isotope effects in glucose by (13)C NMR spectroscopy and computational studies.

Anomeric equilibrium isotope effects for dissolved sugars are required preludes to understanding isotope effects for these molecules bound to enzymes. This paper presents a full molecule study of the alpha- and beta-anomeric forms of D-glucopyranose in water using deuterium conformational equilibrium isotope effects (CEIE). Using 1D (13)C NMR, we have found deuterium isotope effects of 1.043 +/- 0.004, 1.027 +/- 0.005, 1.027 +/- 0.004, 1.001 +/- 0.003, 1.036 +/- 0.004, and 0.998 +/- 0.004 on the equilibrium constant, (H/D)K(beta/alpha), in [1-(2)H]-, [2-(2)H]-, [3-(2)H]-, [4-(2)H]-, [5-(2)H]-, and [6,6'-(2)H(2)]-labeled sugars, respectively. A computational study of the anomeric equilibrium in glucose using semiempirical and ab initio methods yields values that correlate well with experiment. Natural bond orbital (NBO) analysis of glucose and dihedral rotational equilibrium isotope effects in 2-propanol strongly imply a hyperconjugative mechanism for the isotope effects at H1 and H2. We conclude that the isotope effect at H1 is due to n(p) --> sigma* hyperconjugative transfer from O5 to the axial C1--H1 bond in beta-glucose, while this transfer makes no contribution to the isotope effect at H5. The isotope effect at H2 is due to rotational restriction of OH2 at 160 degrees in the alpha form and 60 degrees in the beta-sugar, with concomitant differences in n --> sigma* hyperconjugative transfer from O2 to CH2. The isotope effects on H3 and H5 result primarily from syn-diaxial steric repulsion between these and the axial anomeric hydroxyl oxygen in alpha-glucose. Therefore, intramolecular effects play an important role in isotopic perturbation of the anomeric equilibrium. The possible role of intermolecular effects is discussed in the context of recent molecular dynamics studies on aqueous glucose.     

Quantitative 15N NMR spectroscopy

Line intensities in ¹⁵N NMR spectra are strongly influenced by spin-lattice and spin–spin relaxation times, relaxation mechanisms and experimental conditions. Special care has to be taken in using ¹⁵N spectra for quantitative purposes. Quantitative aspects are discussed for the ¹⁵N NMR of molecules with different nitrogen functional groups and also mixtures of nitrogen-containing compounds. It is shown that, in general, quantitative data are obtainable from integration of ¹⁵N lines in proton decoupled ¹⁵N NMR spectra using NOE suppression. Addition of paramagnetic relaxation reagents (PARR) under controlled conditions is frequently needed to accomplish the experiment within reasonable time limits.     

Functional genomics and NMR spectroscopy

The recent success of the human genome project and the continued accomplishment in obtaining DNA sequences for a vast array of organisms is providing an unprecedented wealth of information. Nevertheless, an abundance of the proteome contains hypothetical proteins or proteins of unknown function, where high throughput approaches for genome-wide functional annotation (functional genomics) has evolved as the necessary next step. Nuclear magnetic resonance spectroscopy is playing an important role in functional genomics by providing information on the structure of protein and protein-ligand complexes, from metabolite fingerprinting and profiling, from the analysis of the metabolome, and from ligand affinity screens to identify chemical probes.