Phosphorylated Peptide Derived from the Myosin Phosphatase Target Subunit Is a Novel Inhibitor of Protein Phosphatase-1.

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R690QSRRS(pT696)QGVTL701 (P-Thr696-MYPT1690-701), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1690-701 was dephosphorylated by PP1c slowly (t1/2 = 81.6-87.9 min), which was further impeded (t1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t1/2 = 1.69 min) significantly (t1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1690-701) or phosphoserine (PP1c-P-Ser696-MYPT1690-701) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.

DMSO-Induced Unfolding of the Antifungal Disulfide Protein PAF and Its Inactive Variant: A Combined NMR and DSC Study.

PAF and related antifungal proteins are promising antimicrobial agents. They have highly stable folds around room temperature due to the presence of 3-4 disulfide bonds. However, unfolded states persist and contribute to the thermal equilibrium in aqueous solution, and low-populated states might influence their biological impact. To explore such equilibria during dimethyl sulfoxide (DMSO)-induced chemical unfolding, we studied PAF and its inactive variant PAFD19S using nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). According to the NMR monitoring at 310 K, the folded structures disappear above 80 v/v% DMSO concentration, while the unfolding is completely reversible. Evaluation of a few resolved peaks from viscosity-compensated 15N-1H HSQC spectra of PAF yielded ∆G = 23 ± 7 kJ/M as the average value for NMR unfolding enthalpy. The NMR-based structures of PAF and the mutant in 50 v/v% DMSO/H2O mixtures were more similar in the mixed solvents then they were in water. The 15N NMR relaxation dynamics in the same mixtures verified the rigid backbones of the NMR-visible fractions of the proteins; still, enhanced dynamics around the termini and some loops were observed. DSC monitoring of the Tm melting point showed parabolic dependence on the DMSO molar fraction and suggested that PAF is more stable than the inactive PAFD19S. The DSC experiments were irreversible due to the applied broad temperature range, but still suggestive of the endothermic unfolding of PAF.

Synthesis of a Heparinoid Pentasaccharide Containing l-Guluronic Acid Instead of l-Iduronic Acid with Preserved Anticoagulant Activity.

l-Iduronic acid is a key constituent of heparin and heparan sulfate polysaccharides due to its unique conformational plasticity, which facilitates the binding of polysaccharides to proteins. At the same time, this is the synthetically most challenging unit of heparinoid oligosaccharides; therefore, there is a high demand for its replacement with a more easily accessible sugar unit. In the case of idraparinux, an excellent anticoagulant heparinoid pentasaccharide, we demonstrated that l-iduronic acid can be replaced by an easier-to-produce l-sugar while maintaining its essential biological activity. From the inexpensive d-mannose, through a highly functionalized phenylthio mannoside, the l-gulose donor was prepared by C-5 epimerization in 10 steps with excellent yield. This unit was incorporated into the pentasaccharide by α-selective glycosylation and oxidized to l-guluronic acid. The complete synthesis required only 36 steps, with 21 steps for the longest linear route. The guluronate containing pentasaccharide inhibited coagulation factor Xa by 50% relative to the parent compound, representing an excellent anticoagulant activity. To the best of our knowledge, this is the first biologically active heparinoid anticoagulant which contains a different sugar unit instead of l-iduronic acid.

A Fucosylated Lactose-Presenting Tetravalent Glycocluster Acting as a Mutual Ligand of Pseudomonas aeruginosa Lectins A (PA-IL) and B (PA-IIL)-Synthesis and Interaction Studies.

The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic human pathogen associated with cystic fibrosis. P. aeruginosa produces two soluble lectins, the d-galactose-specific lectin PA-IL (LecA) and the l-fucose-specific lectin PA-IIL (LecB), among other virulence factors. These lectins play an important role in the adhesion to host cells and biofilm formation. Moreover, PA-IL is cytotoxic to respiratory cells in the primary culture. Therefore, these lectins are promising therapeutic targets. Specifically, carbohydrate-based compounds could inhibit their activity. In the present work, a 3-O-fucosyl lactose-containing tetravalent glycocluster was synthesized and utilized as a mutual ligand of galactophilic and fucophilic lectins. Pentaerythritol equipped with azido ethylene glycol-linkers was chosen as a multivalent scaffold and the glycocluster was constructed by coupling the scaffold with propargyl 3-O-fucosyl lactoside using an azide-alkyne 1,3-dipolar cycloaddition reaction. The interactions between the glycocluster and PA-IL or PA-IIL were investigated by isothermal titration microcalorimetry and saturation transfer difference NMR spectroscopy. These results may assist in the development of efficient anti-adhesion therapy for the treatment of a P. aeruginosa infection.

Expedited Nuclear Magnetic Resonance Assignment of Small- to Medium-Sized Molecules with Improved HSQC-CLIP-COSY Experiments.

Resonance assignment is a pivotal step for any nuclear magnetic resonance (NMR) analysis, such as structure elucidation or the investigation of protein-ligand interactions. Both 1H-13C heteronuclear single quantum correlation (HSQC) and 1H-1H correlation spectroscopy (COSY) two-dimensional (2D) experiments are invaluable for 1H NMR assignment, by extending the high signal dispersion of 13C chemical shifts onto 1H resonances and by providing a high amount of through-bond 1H-1H connectivity information, respectively. The recently introduced HSQC-CLIP(Clean In-Phase)-COSY method combines these two experiments, providing COSY correlations along the high-resolution 13C dimension with clean in-phase multiplets. However, two experiments need to be recorded to unambiguously identify COSY cross-peaks. Here, we propose novel variants of the HSQC-CLIP-COSY pulse sequence that edit cross-peak signs so that direct HSQC responses can be distinguished from COSY relay peaks, and/or the multiplicities of the 13C nuclei are reflected, allowing the assignment of all the peaks in a single experiment. The advanced HSQC-CLIP-COSY variants have the potential to accelerate and simplify the NMR structure-elucidation process of both synthetic and natural products and to become valuable tools for high-throughput computer-assisted structure determination.

Conformational Analysis of Heparin-Analogue Pentasaccharides by Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulations.

Elucidation and improvement of the blood coagulant properties of heparin are the focus of intense research. In this study, we performed conformational analysis using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations on the heparin pentasaccharide analogue idraparinux, its disulfonatomethyl analogue, which features a slightly improved blood coagulation property, and a trisulfonatomethyl analogue, in which the activity has been totally abolished. As the ring conformation of the G subunit has been suggested as a major determinant of the biological properties, we analyzed the sugar ring conformations and dynamics of the interglycosidic linkages. We found that the conformation of the G ring is dominated by the 2SO skewed boat next to the 1C4 chair in all three derivatives. Both the thermodynamics and the kinetics of the conformational states were found to be highly similar in the three derivatives. Molecular kinetic analysis showed that the 2SO skewed boat state of the G ring is equally favorable in the three analogues, resulting in similar 2SO populations. Also, the transition kinetics from the 1C4 chair to the 2SO skewed boat was found to be comparable in the derivatives, which indicates a similar energy barrier between the two states of the G subunit. We also identified a slower conformational transition between the dominant 4C1 chair and the boat conformations on the E subunit. Both G and E ring flips are also accompanied by changes along the interglycosidic linkages, which take place highly synchronously with the ring flips. These findings indicate that conformational plasticity of the G ring and the dominance of the 2SO skewed boat populations do not necessarily warrant the biological activity of the derivatives and hence the impact of other factors also needs to be considered.

Synthesis of Tetravalent Thio- and Selenogalactoside-Presenting Galactoclusters and Their Interactions with Bacterial Lectin PA-IL from Pseudomonas aeruginosa.

Synthesis of tetravalent thio- and selenogalactopyranoside-containing glycoclusters using azide-alkyne click strategy is presented. Prepared compounds are potential ligands of Pseudomonas aeruginosa lectin PA-IL. P. aeruginosa is an opportunistic human pathogen associated with cystic fibrosis, and PA-IL is one of its virulence factors. The interactions of PA-IL and tetravalent glycoconjugates were investigated using hemagglutination inhibition assay and compared with mono- and divalent galactosides (propargyl 1-thio- and 1-seleno-ß-d-galactopyranoside, digalactosyl diselenide and digalactosyl disulfide). The lectin-carbohydrate interactions were also studied by saturation transfer difference NMR technique. Both thio- and seleno-tetravalent glycoconjugates were able to inhibit PA-IL significantly better than simple d-galactose or their intermediate compounds from the synthesis.

NORD: NO Relaxation Delay NMR Spectroscopy.

The novel concept of NORD (NO relaxation delay) NMR spectroscopy is introduced. The idea is to design concatenated experiments in a way that the magnetization used in the first relaxes toward equilibrium during the second and vice versa, thus saving instrument time. Applications include complete well-resolved 1 H-1 H and 1 H-13 C one-bond and long-range correlation maps of an 80 mM solution of a trisaccharide recorded in less than two minutes and hydrocortisone with extensive spectral overlap.

Pigments of the Moss Paraleucobryum longifolium: Isolation and Structure Elucidation of Prenyl-Substituted 8,8'-Linked 9,10-Phenanthrenequinone Dimers.

In a search for new secondary metabolites from mosses, leucobryns A-E, axially chiral 9,10-phenanthrenequinone dimers, were isolated from Paraleucobryum longifolium (1-5), together with diosmetin triglycoside. Leucobryns B (2) and C (3) were proved to be homodimeric atropodiastereomers containing both axial and central chirality elements, while leucobryns D (4) and E (5) were found to be heterodimeric atropodiastereomers containing central chirality in only one of the two monomeric units. Axial chirality of the compounds was determined by ECD measurements and sTDA ECD calculations, while the central chirality elements were assigned by TDDFT-SOR calculations. Leucobryns represent the first 9,10-phenanthrenequinone dimers, the monomers of which are linked through their C-8 atoms. Leucobryns B-E contain an uncommon C10 monoterpenoid side chain, in which isoprenoid units are joined by 3,4 linkages. Leucobryns A and B exhibited weak antiproliferative activity against several human cancer cell lines.

Selenoglycosides as Lectin Ligands: 77 Se-Edited CPMG-HSQMBC NMR Spectroscopy To Monitor Biomedically Relevant Interactions.

The fundamental importance of protein-glycan recognition calls for specific and sensitive high-resolution techniques for their detailed analysis. After the introduction of 19 F NMR spectroscopy to study the recognition of fluorinated glycans, a new 77 Se NMR spectroscopy method is presented for complementary studies of selenoglycans with optimised resolution and sensitivity, in which direct NMR spectroscopy detection on 77 Se is replaced by its indirect observation in a 2D 1 H,77 Se HSQMBC spectrum. In contrast to OH/F substitution, O/Se exchange allows the glycosidic bond to be targeted. As an example, selenodigalactoside recognition by three human galectins and a plant toxin is readily indicated by signal attenuation and line broadening in the 2D 1 H,77 Se HSQMBC spectrum, in which CPMG-INEPT long-range transfer ensures maximal detection sensitivity, clean signal phases, and reliable ligand ranking. By monitoring competitive displacement of a selenated spy ligand, the selective 77 Se NMR spectroscopy approach may also be used to screen non-selenated compounds. Finally, 1 H,77 Se CPMG-INEPT transfer allows further NMR sensors of molecular interaction to be combined with the specificity and resolution of 77 Se NMR spectroscopy.

Comparison of Carbohydrate Force Fields Using Gaussian Accelerated Molecular Dynamics Simulations and Development of Force Field Parameters for Heparin-Analogue Pentasaccharides.

Computational description of conformational and dynamic properties of anticoagulant heparin analogue pentasaccharides is of crucial importance in understanding their biological activities. We designed and synthesized idraparinux derivatives modified with sulfonatomethyl moieties at the D, F, and H glucose units that display varied potencies depending on the exact nature of the substitution. In this report we examined the capability of molecular dynamics (MD) simulations to describe the conformational behavior of these novel idraparinux derivatives. We used Gaussian accelerated MD (GAMD) simulations on the parent compound, idraparinux, to choose the most suitable carbohydrate force field for these type of compounds. GAMD provided significant acceleration of conformational transitions compared to classical MD. We compared descriptors obtained from GAMD with NMR spectroscopic parameters related to geometrical descriptors such as scalar couplings and nuclear Overhauser effects (NOE) measured on idraparinux. We found that the experimental data of idraparinux is best reproduced by the CHARMM carbohydrate force field. Furthermore, we propose a torsion angle parameter for the sulfonato-methyl group, which was developed for the chosen CHARMM force field using quantum chemical calculations and validated by comparison with NMR data. The work lays down the foundation of using MD simulations to gain insight into the conformational properties of sulfonato-methyl group modified idraparinux derivatives and to understand their structure-activity relationship thus enabling rational design of further modifications.

Inhibition of protein phosphatase-1 and -2A by ellagitannins: structure-inhibitory potency relationships and influences on cellular systems.

Several ellagitannins inhibited the activity of protein phosphatase-1 (PP1) and -2 A (PP2A) catalytic subunits (PP1c and PP2Ac) with preferential suppression of PP1c over PP2Ac. The inhibitory potency for PP1c followed the order of tellimagrandin I > mahtabin A > praecoxin B > 1.2-Di-O-galloyl-4.6-(S)-HHDP-ß-D-glucopyranose > pedunculagin with IC50 values ranging from 0.20 µM to 2.47 µM. The interaction of PP1c and tellimagrandin I was assessed by NMR saturation transfer difference, surface plasmon resonance, isothermal titration calorimetry, and microscale thermophoresis based binding techniques. Tellimagrandin I suppressed viability and phosphatase activity of HeLa cells, while mahtabin A was without effect. Conversely, mahtabin A increased the phosphorylation level of SNAP-25Thr138 and suppressed exocytosis of cortical synaptosomes, whereas tellimagrandin I was without influence. Our results establish ellagitannins as partially selective inhibitors of PP1 and indicate that these polyphenols may act distinctly in cellular systems depending on their membrane permeability and/or their actions on cell membranes.

Synthesis of α-l-Fucopyranoside-Presenting Glycoclusters and Investigation of Their Interaction with Photorhabdus asymbiotica Lectin (PHL).

Photorhabdus asymbiotica is a gram-negative bacterium that is not only as effective an insect pathogen as other members of the genus, but it also causes serious diseases in humans. The recently identified lectin PHL from P. asymbiotica verifiably modulates an immune response of humans and insects, which supports the idea that the lectin might play an important role in the host-pathogen interaction. Dimeric PHL contains up to seven l-fucose-specific binding sites per monomer, and in order to target multiple binding sites of PHL, α-l-fucoside-containing di-, tri- and tetravalent glycoclusters were synthesized. Methyl gallate and pentaerythritol were chosen as multivalent scaffolds, and the fucoclusters were built from the above-mentioned cores by coupling with different oligoethylene bridges and propargyl α-l-fucosides using 1,3-dipolar azide-alkyne cycloaddition. The interaction between fucoside derivates and PHL was investigated by several biophysical and biological methods, ITC and SPR measurements, hemagglutination inhibition assay, and an investigation of bacterial aggregation properties were carried out. Moreover, details of the interaction between PHL and propargyl α-l-fucoside as a monomer unit were revealed using X-ray crystallography. Besides this, the interaction with multivalent compounds was studied by NMR techniques. The newly synthesized multivalent fucoclusters proved to be up to several orders of magnitude better ligands than the natural ligand, l-fucose.

Aralkyl selenoglycosides and related selenosugars in acetylated form activate protein phosphatase-1 and -2A.

Aralkyl and aryl selenoglycosides as well as glycosyl selenocarboxylate derivatives were assayed on the activity of protein phosphatase-1 (PP1) and -2A (PP2A) catalytic subunits (PP1c and PP2Ac) in search of compounds for PP1c and PP2Ac effectors. The majority of tested selenoglycosides activated both PP1c and PP2Ac by ∼2-4-fold in a phosphatase assay with phosphorylated myosin light chain substrate when the hydroxyl groups of the glycosyl moiety were acetylated, but they were without any effects in the non-acetylated forms. A peptide from the myosin phosphatase target subunit-1 (MYPT123-38) that included an RVxF PP1c-binding motif attenuated activation of PP1c by 2-Trifluoromethylbenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-ß-d-glucopyranoside (TFM-BASG) and 4-Bromobenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-ß-d-glucopyranoside (Br-BASG). MYPT123-38 stimulated PP2Ac and contributed to PP2Ac activation exerted by either Br-BASG or TFM-BASG. Br-BASG and TFM-BASG suppressed partially binding of PP1c to MYPT1 in surface plasmon resonance based binding experiments. Molecular docking predicted that the hydrophobic binding surfaces in PP1c for interaction with either the RVxF residues of PP1c-interactors or selenoglycosides are partially overlapped. Br-BASG and TFM-BASG caused a moderate increase in the phosphatase activity of HeLa cells in 1 h, and suppressed cell viability in 24 h incubations. In conclusion, our present study identified selenoglycosides as novel activators of PP1 and PP2A as well as provided insights into the structural background of their interactions establishing a molecular model for future design of more efficient phosphatase activator molecules.

Broadband homonuclear decoupled HSQMBC methods.

Long-range heteronuclear coupling constants convey invaluable information for stereochemical and conformational analysis of molecules from synthetic and natural origin. Here, we report a real-time Zangger-Sterk CPMG-HSQMBC method for the precise and direct measurement of multiple-bond heteronuclear couplings. It is demonstrated that the real-time acquisition strategy combined with multiple slice selective excitation can provide substantial improvement in sensitivity (or reduction of experimental time) as compared to other variants of broadband homonuclear decoupled HSQMBC methods published previously. Scope and limitations of the different strategies applied for decoupling are reviewed. Moreover, practical guidelines for the choice of the most appropriate method are also presented. Applications are given on a metal complex incorporating P-heterocycles and two diglycosyl-selenides for the extraction of n J(1 H, 31 P) and n J(1 H, 77 Se), respectively.

Photochemical and Structural Studies on Cyclic Peptide Models.

Ultra-violet (UV) irradiation has a significant impact on the structure and function of proteins that is supposed to be in relationship with the tryptophan-mediated photolysis of disulfide bonds. To investigate the correlation between the photoexcitation of Trp residues in polypeptides and the associated reduction of disulfide bridges, a series of small, cyclic oligopeptide models were analyzed in this work. Average distances between the aromatic side chains and the disulfide bridge were determined following molecular mechanics (MM) geometry optimizations. In this way, the possibility of cation⁻π interactions was also investigated. Molecular mechanics calculations revealed that the shortest distance between the side chain of the Trp residues and the disulfide bridge is approximately 5 Å in the cyclic pentapeptide models. Based on this, three tryptophan-containing cyclopeptide models were synthesized and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Experimental data and detailed molecular dynamics (MD) simulations were in good agreement with MM geometry calculations. Selected model peptides were subjected to photolytic degradation to study the correlation of structural features and the photolytic cleavage of disulfide bonds in solution. Formation of free sulfhydryl groups upon illumination with near UV light was monitored by fluorescence spectroscopy after chemical derivatization with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) and mass spectrometry. Liquid cromatography-mass spectrometry (LC-MS) measurements indicated the presence of multiple photooxidation products (e.g., dimers, multimers and other oxidated products), suggesting that besides the photolysis of disulfide bonds secondary photolytic processes take place.

Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR.

Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used 31P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and 31P T2 relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T2 relaxation are observed at higher temperatures. A comparison of 31P T1 values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T1 relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules--in particular for the negatively charged DMPG--while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide-membrane interactions.

Target-specific NMR detection of protein-ligand interactions with antibody-relayed 15N-group selective STD.

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein-ligand interactions is a key element. 1H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

Real-time pure shift ¹5N HSQC of proteins: a real improvement in resolution and sensitivity.

Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t 2) broadband homodecoupling, suppressing the multiplet structure caused by proton-proton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to (15)N, while the former selects a region of the (1)H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments.

PSYCHE CPMG-HSQMBC: An NMR Spectroscopic Method for Precise and Simple Measurement of Long-Range Heteronuclear Coupling Constants.

Among the NMR spectroscopic parameters, long-range heteronuclear coupling constants convey invaluable information on torsion angles relevant to glycosidic linkages of carbohydrates. A broadband homonuclear decoupled PSYCHE CPMG-HSQMBC method for the precise and direct measurement of multiple-bond heteronuclear couplings is presented. The PSYCHE scheme built into the pulse sequence efficiently eliminates unwanted proton-proton splittings from the heteronuclear multiplets so that the desired heteronuclear couplings can be determined simply by measuring frequency differences between peak maxima of pure antiphase doublets. Moreover, PSYCHE CPMG-HSQMBC can provide significant improvement in sensitivity as compared to an earlier Zangger-Sterk-based method. Applications of the proposed pulse sequence are demonstrated for the extraction of (n)J((1)H,(77)Se) and (n)J((1)H,(13)C) values, respectively, in carbohydrates; further extensions can be envisioned in any J-based structural and conformational studies.