Heteronuclear Multiple-quantum Coherence Experiments Research Articles

35 Cl-1 H Heteronuclear correlation magic-angle spinning nuclear magnetic resonance experiments for probing pharmaceutical salts.

Heteronuclear multiple-quantum coherence (HMQC) pulse sequences for establishing heteronuclear correlation in solid-state nuclear magnetic resonance (NMR) between 35 Cl and 1 H nuclei in chloride salts under fast (60 kHz) magic-angle spinning (MAS) and at high magnetic field (a 1 H Larmor frequency of 850 MHz) are investigated. Specifically, recoupling of the 35 Cl-1 H dipolar interaction using rotary resonance recoupling with phase inversion every rotor period or the symmetry-based SR42 1 pulse sequences are compared. In our implementation of the population transfer (PT) dipolar (D) HMQC experiment, the satellite transitions of the 35 Cl nuclei are saturated with an off-resonance WURST sweep, at a low nutation frequency, over the second spinning sideband, whereby the WURST pulse must be of the same duration as the recoupling time. Numerical simulations of the 35 Cl-1 H MAS D-HMQC experiment performed separately for each crystallite orientation in a powder provide insight into the orientation dependence of changes in the second-order quadrupolar-broadened 35 Cl MAS NMR lineshape under the application of dipolar recoupling. Two-dimensional 35 Cl-1 H PT-D-HMQC MAS NMR spectra are presented for the amino acids glycine·HCl and l-tyrosine·HCl and the pharmaceuticals cimetidine·HCl, amitriptyline·HCl and lidocaine·HCl·H2 O. Experimentally observed 35 Cl lineshapes are compared with those simulated for 35 Cl chemical shift and quadrupolar parameters as calculated using the gauge-including projector-augmented wave (GIPAW) method: the calculated quadrupolar product (PQ ) values exceed those measured experimentally by a factor of between 1.3 and 1.9.

Fast Acquisition of Proton-Detected HETCOR Solid-State NMR Spectra of Quadrupolar Nuclei and Rapid Measurement of NH Bond Lengths by Frequency Selective HMQC and RESPDOR Pulse Sequences.

Fast magic-angle spinning (MAS), frequency selective (FS) heteronuclear multiple quantum coherence (HMQC) experiments which function in an analogous manner to solution SOFAST HMQC NMR experiments, are demonstrated. Fast MAS enables efficient FS excitation of 1 H solid-state NMR signals. Selective excitation and observation preserves 1 H magnetization, leading to a significant shortening of the optimal inter-scan delay. Dipolar and scalar 1 H{14 N} FS HMQC solid-state NMR experiments routinely provide 4- to 9-fold reductions in experiment times as compared to conventional 1 H{14 N} HMQC solid-state NMR experiments. 1 H{14 N} FS resonance-echo saturation-pulse double-resonance (RESPDOR) allowed dipolar dephasing curves to be obtained in minutes, enabling the rapid determination of NH dipolar coupling constants and internuclear distances. 1 H{14 N} FS RESPDOR was used to assign multicomponent active pharmaceutical ingredients (APIs) as salts or cocrystals. FS HMQC also provided enhanced sensitivity for 1 H{17 O} and 1 H{35 Cl} HMQC experiments on 17 O-labeled Fmoc-alanine and histidine hydrochloride monohydrate, respectively. FS HMQC and FS RESPDOR experiments will provide access to valuable structural constraints from materials that are challenging to study due to unfavorable relaxation times or dilution of the nuclei of interest.

Simple and Efficient Synthesis of Novel 3‐Substituted 2‐Thioxo‐2,3‐dihydro‐1H‐benzo[g]quinazolin‐4‐ones and Their Reactions with Alkyl Halides and α‐Glycopyranosyl Bromides

A series of 3‐substituted 2‐thioxo‐2,3‐dihydro‐1H‐benzo[g]quinazolin‐4‐ones 4a–e were synthesized from the reaction of 3‐aminonaphthalene‐2‐carboxylic acid 1 with isothiocyanate derivatives 2a–e. The alkylation of 4a–e with alkyl halides gave 3‐substituted 2‐alkylsulfanyl‐2,3‐dihydro‐1H‐benzo[g]quinazolin‐4‐ones 5a–o. S‐Glycosylation was carried out via the reaction of 4a–e with glycopyranosyl bromides 7a and 7b under anhydrous alkaline conditions. The structure of the compounds was established as S‐nucleoside and not N‐nucleoside. Conformational analysis has been studied by homonuclear and heteronuclear two‐dimensional NMR methods (2D DFQ‐COSY, heteronuclear multiple quantum coherence, and heteronuclear multiple bond correlation). The S site of alkylation and glycosylation was determined from the 1H and 13C heteronuclear multiple quantum coherence experiments.

Characterization of Chemical Constituents of <i>Luffa operculata</i>(Cucurbitaceae)

A mixture of new ceramides (1, 2, 3, 4 and 5) together with a binary mixture of ceramides with long chain alkyl (6 and 7), triterpenoid (10) and steroids (11 and 12) have been isolated from bark of the fruits and of the stems of Luffa operculata (Cucurbitaceae). The structures were elucidated by comprehensive spectro- scopic analysis including 1H and 13C NMR, DEPT (distortionless enhancement by polarization transfer), COSY (correlated spectroscopy), HMQC (heteronuclear multiple quantum coherence), HMBC (heteronu- clear multiple bond connectivity), IR (infrared), HR-ESI-MS (electrospray ionization-high resolution mass spectra) and LR-MS (low resolution electron ionization mass spectra) experiments. All the ceramides are reported for the first time in Cucurbitaceae and this is the first report of the rare triterpene 10 isolated from Luffa operculata. The ceramides 6 and 7 showed a high acetylcholine esterase inhibitory effect.

Determining the Charge State of Histidine Side Chains in Antimicrobial Piscidin By Nuclear Magnetic Resonance

Piscidins constitute a family of three antimicrobial peptides discovered in the mast cells of hybrid striped bass. These peptides, which are highly cationic, contain several arginine and histidine residues. While piscidin 1 is the most antimicrobial and hemolytic isoform, piscidin 3, which has slightly lower antimicrobial activity, is significantly less hemolytic. One of the most striking differences between piscidin 1 and 3 is the substitution of glycine for the histidine at position 17 in piscidin 1.As part of its mechanism of action, piscidin recognizes negatively charged microbial membranes. Therefore, studying the interactions of the piscidin with lipids can help us better understand the chemical basis of its antimicrobial and hemolytic effects. Because physiological pH is around 7.4, and the average pKa of histidine side chains is around 6.0, a detailed study of the histidine side chains in piscidin 1 and 3 is needed to discern the charge state of the peptides under physiological conditions. In this research, we used solution nuclear magnetic resonance to obtain the pKa of the histidine side chains of piscidin bound to sodium dodecyl sulfate micelles. Heteronuclear multiple quantum coherence experiments were performed on piscidin 1 and 3 containing 15N-side chain labeled histidines. 15N and 1H chemical shifts were recorded as a function of pH to determine the titration curve of each histidine residue. The results will be discussed in the context of structure-function relationships in membrane-active peptides. The knowledge gained from these studies can help identify common principles that will facilitate the design of pharmaceuticals with broad-spectrum antibacterial activity, minimum induction of bacterial resistance, and low toxicity to mammalian cells.

A Complete Product Operator Theory for IS (I=1, S = 1) Spin System and Application to 3D HMQC-COSY NMR Experiment

There exist a variety of multi-pulse NMR experiments for spectral assignment of complex molecules in solution. The conventional heteronuclear multiple-quantum coherence (HMQC) NMR experiment provides correlation between weakly coupled hetero-nuclei. The COSY is one of the most popular two-dimensional NMR experiment which is used to correlate J-coupled homo-nuclei of spectral assignment. The combination of the conventional HMQC and COSY NMR experiments yields a new experiment called 3D HMQC-COSY NMR experiment. The product operator theory is widely used for the analytical descriptions of multi-pulse NMR experiments for weakly coupled spin systems in liquids. In this study, complete product operator theory for weakly coupled IS (I = 1, S = 1) spin system is presented by obtaining the evolutions of the product operators under the spin-spin coupling Hamiltonian. As an application and a verification, analytical descriptions of 3D HMQC-COSY NMR experiment are obtained for weakly coupled ISnI’_S’m (I = I’ = 1/2; S = S’ = 1; n = 1,2, 3; m = 1, 2) multi-spin systems. Then the estimated spectra of this experiment for various multi-spin systems are explained in detail.

Fluxionality in a Paramagnetic Seven-Coordinate Iron(II) Complex: A Variable-Temperature, Two-Dimensional NMR and DFT Study

The preparation and detailed characterizations of the high-spin seven-coordinate complexes [M(kappa(7)N-L)](ClO(4))(2) (M = Mn(II), Fe(II); L = N,N,N',N'-tetrakis(2-pyridylmethyl)-2,6-bis(aminomethyl)pyridine) are described. The X-ray crystal structures reveal seven-coordinate metal complex ions. Consideration of continuous shape measures reveals that the coordination environments about the metal ions are better described as having (C(s)) face-capped trigonal prismatic symmetry than (C(2)) pentagonal bipyramidal symmetry. The (S = (5)/(2)) Mn(II) species shows complicated X-band electron paramagnetic resonance (EPR) spectra and broad, unrevealing (1)H NMR spectra. In contrast, the (S = 2) Fe(II) complex is EPR-silent and shows completely interpretable (1)H NMR spectra containing the requisite number of paramagnetically shifted peaks in the range delta +150 to -60. The (13)C NMR spectra are likewise informative. Variable-temperature (1)H NMR spectra show coalescences and decoalescences indicative of an intramolecular process that pairwise-exchanges the nonequivalent pyridylmethyl "arms" of the two bis(pyridylmethyl)amine (bpa) domains. A suite of NMR techniques, including T(1) relaxation measurements and variable-temperature (1)H-(1)H correlation spectroscopy, (1)H-(1)H total correlation spectroscopy, (1)H-(1)H nuclear Overhauser effect spectroscopy/exchange spectroscopy, and (1)H-(13)C heteronuclear multiple-quantum coherence experiments, has been used to assign the NMR spectra and characterize the exchange process. Analysis of the data from these experiments yields the following thermodynamic parameters for the exchange: DeltaH++ = 53.6 +/- 2.8 kJ mol(-1), DeltaS++ = -10.0 +/- 9.8 J K(-1) mol(-1), and DeltaG++ (298 K) = 50.6 kJ mol(-1). Density functional theory (B3LYP) calculations have been used to explore the energetics of possible mechanistic pathways for the underlying fluxional process. The most plausible mechanism found involves dissociation of a pyridylmethyl arm to afford a strained six-coordinate species followed by rebinding of the arm in a different position to afford a new seven-coordinate transition state in which the pyridylmethyl arms within each bpa domain are essentially equivalent; the calculated energy barrier for this process is 53.5 kJ mol(-1), in good agreement with the observations.

Very Fast Two-Dimensional NMR Spectroscopy for Real-Time Investigation of Dynamic Events in Proteins on the Time Scale of Seconds

We demonstrate for different protein samples that 2D 1H-15N correlation NMR spectra can be recorded in a few seconds of acquisition time using a new band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence experiment. This has enabled us to measure fast hydrogen-deuterium exchange rate constants along the backbone of a small globular protein fragment by real-time 2D NMR.

Binding of Two Flaviolin Substrate Molecules, Oxidative Coupling, and Crystal Structure of Streptomyces coelicolor A3(2) Cytochrome P450 158A2

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

Coordination of aluminium with purpurogallin and theaflavin digallate

Polyphenols are antioxidants, which are known to influence bioavailability of metals in the body. The theaflavins of black tea are important members of this family, which have been sparsely investigated. The complexation of aluminium with purpurogallin (2,3,4,6-tetrahydroxy-5H-benzocyclohepten-5-one) has been investigated using nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography–mass spectroscopy (LC–MS) and Fourier transform infrared (FT-IR) spectroscopy. 1H NMR was used to determine the coordination site of the aluminium ion and LC–MS to determine the stoichiometry and molecular weight of the major complex formed in solution. FT-IR spectral comparisons were used to corroborate the proposed chelating moiety. The complexation of aluminium with the high-molecular-weight, tea polyphenol theaflavin digallate was also investigated using 1H NMR and heteronuclear multiple quantum coherence experiments. Structures of the major aluminium purpurogallin and aluminium theaflavin digallate complexes are proposed.

Reconstructing NMR spectra of "invisible" excited protein states using HSQC and HMQC experiments.

Carr-Purcell-Meiboom-Gill (CPMG) relaxation measurements employing trains of 180 degrees pulses with variable pulse spacing provide valuable information about systems undergoing millisecond-time-scale chemical exchange. Fits of the CPMG relaxation dispersion profiles yield rates of interconversion, relative populations, and absolute values of chemical shift differences between the exchanging states, |Deltaomega|. It is shown that the sign of Deltaomega that is lacking from CPMG dispersion experiments can be obtained from a comparison of chemical shifts in the indirect dimensions in either a pair of HSQC (heteronuclear single quantum coherence) spectra recorded at different magnetic fields or HSQC and HMQC (heteronuclear multiple quantum coherence) spectra obtained at a single field. The methodology is illustrated with an application to a cavity mutant of T4 lysozyme in which a leucine at position 99 has been replaced by an alanine, giving rise to exchange between ground state and excited state conformations with a rate on the order of 1450 s(-1) at 25 degrees C.

THE APPLICATION OF MULTIDIMENSIONAL NMR TO THE STUDY OF SOIL HUMIC SUBSTANCES

Humic substances are the most abundant organic macromolecules in soils, and comprehension of their chemical structure is essential to understanding their role in terrestrial ecosystems. The one-dimensional nuclear magnetic resonance (NMR) spectroscopy techniques now used widely to study humic substances have provided important insight into humic structures, but the complexity of these macromolecules gives rise to resonance signals that are broad and have spectral overlap. This has prevented the definitive functional group assignments necessary for structural determination. Hence, interest has focused on more powerful two-dimensional NMR experiments, such as the homonuclear TOtal Correlation SpectroscopY (TOCSY) and Heteronuclear Multiple Quantum Coherence (HMQC), which were employed in the study of a soil humic acid standard. The purpose of this paper is to outline the potential of these techniques to the study of soil humic structures. The 2-D spectra produced were extremely encouraging, with multitudes of cross-peaks produced from both TOCSY and HMQC experiments. The identification of fatty ester/acid chains and amino acid couplings are given as examples. Results obtained with these NMR experiments indicate substantial improvements in functional group assignment capabilities and the potential for marked progress in the determination of the chemical structure of soil humic substances.

Simulation of the off-resonance effect in one-dimensional heteronuclear multiple quantum coherence spectroscopy

When a heteronuclear multiple quantum coherence (HMQC) NMR experiment is performed in one-dimensional mode, due to the wide range in chemical shift of the indirectly detected spin and the limited strength of the radio-frequency field, the off-resonance effect on the intensity of the observed signal can be serious. In this paper, the effect is studied using the spin-density-matrix formalism and simulations of the experimental results are presented. The bilinear rotation-decoupling sequence (BIRD), which is usually used in HMQC experiments, is also discussed. It is shown that the BIRD sequence has a negative effect by virtue of narrowing the excitation band.

Tryptophan glycoconjugates in food and human urine

Evaluating the formation of tryptophan glycoconjugates other than well-established Amadori rearrangement products, HPLC-tandem MS (MS/MS) analysis of human urine collected from several healthy individuals proved the presence of one distinct tryptophan C-glycosyl compound [Horiuchi, Yonekawa, Iwahara, Kanno, Kurihara and Fujise (1994) J. Biochem. (Tokyo) 115, 362-366]. After isolation, unambiguous identification of this novel tryptophan metabolite as 2-(alpha-mannopyranosyl)-l-tryptophan was achieved by tandem MS combined with NMR spectroscopy including homonuclear COSY, heteronuclear multiple-bond connectivity and (1)H-detected heteronuclear multiple-quantum coherence experiments. Remarkably, a thorough evaluation of vicinal proton-proton coupling constants in different solvents and nuclear Overhauser effect experiments demonstrate the predominant axial orientation of the hydroxymethyl group of the hexopyranosyl residue. Likewise this spatial arrangement indicates that the respective alpha-anomeric C-mannosylhexopyranose is preferentially adopting a (1)C(4) conformation in acidic methanol. Whereas only one distinct tryptophan mannoconjugate could be observed in human urine, HPLC-MS/MS analysis of food samples for the first time led to the identification of numerous N(1)-(beta-d-hexopyranosyl)-l-tryptophan, 2-(beta-d-hexopyranosyl)-l-tryptophan and 1-(1,2,3,4,5-pentahyd- roxypent-1-yl)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid derivatives derived from the condensation of tryptophan with aldohexoses. Taking into consideration the significant differences between profiles and configurations of tryptophan glycoconjugates originating from dietary sources and human urine, C-2 mannosylation of tryptophan residues [de Beer, Vliegenthart, Loeffler and Hofsteenge (1995) Biochemistry 34, 11785-11789] represents a novel enzymic pathway in tryptophan metabolism in humans.

Isolation and characterization of cytochrome c2 from Rhodopseudomonas palustris

Cytochrome c 2 from Rhodopseudomonas palustris has been isolated and characterized by UV-Vis, electron paramagnetic resonance and NMR spectroscopy. The latter, through natural abundance 1H- 13C heteronuclear multiple quantum coherence experiments and homonuclear 2D spectra, allowed us to assign the signals of the heme substituents and of the iron-bound histidine and methionine. An analysis of the 13C chemical shift of the heme substituents has allowed us to localize the axes of the magnetic susceptibility tensor. The latter are consistent with the orientation of the two axial ligands s found from our NOE datae. he pH dependence of the shifts showed a p K a = 6.9 and the presence of a further p K a above 8.0. The present investigation shows how to appropriate use of NMR spectroscopy can provide a wealth of structural and electronic information even in the absence of any biochemical data.

Organic Solvent Systems for31P Nuclear Magnetic Resonance Analysis of Lecithin Phospholipids: Applications to Two-Dimensional Gradient-Enhanced1H-Detected Heteronuclear Multiple Quantum Coherence Experiments

31P NMR of lipid extracts is a reproducible, rapid, and nondegradative method for qualitative and quantitative analyses of phospholipid mixtures. This analysis, however, is hampered by the instability of the solvent system commonly used for NMR spectroscopy (CHCl3/CH3OH/H2O–EDTA). In this work we have investigated the effects of several monophasic solvent mixtures to overcome this disadvantage. Among these mixtures we have selected a solution of triethylamine, dimethylformamide, and guanidinium chloride (Et3N/DMF–GH+) as the most efficient system. In this solvent the chemical shift dispersion of the31P signals is about four times the frequency range observed in the standard chloroform–methanol–water system. Moreover, the stability of this solvent, as a monophasic system, allows easy reproducibility of the analysis. The use of two-dimensional1H–31P gradient-enhanced heteronuclear multiple quantum coherence experiments can further exploit the higher resolution of the signals obtained with this solvent system for the structure elucidation of known and unidentified phospholipids.

Characterization of the glycolipid associated with Alzheimer paired helical filaments.

In the present study, analytical techniques including gas chromatography/mass spectrometry (GC/MS)-assisted carbohydrate linkage-analysis, one- and two-dimensional NMR, and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-MS) have been used to characterize the structure of the glycolipid associated with the paired helical filaments (PHF) isolated from the neurofibrillary tangles of Alzheimer's diseased brain. The 1H NMR spectrum of acid-hydrolyzed protein-resistant core PHF (prcPHF) displays resonances that can be assigned to fatty acid and glucose. There are no resonances present that would indicate the presence of protein, amino acids, or a sphingosine base. Using two-dimensional homonuclear correlated spectroscopy, homonuclear Hartmann-Hahn, and heteronuclear multiple quantum coherence experiments, resonances in the 1H and 13C NMR spectrum of native PHF were assigned to a nonreducing terminal alpha-1,6-glycosidically linked glucose, an internal alpha-1,6-linked glucose, and an alpha-1,2,6-linked glucose. The narrow line-widths observed for these residues suggest that they arise from glucose residues undergoing rapid segmental motion. The carbohydrate portion of the PHF-associated glycolipid was analyzed using GC/MS linkage analysis and confirmed the presence of terminal and internal alpha-1,6-linked glucose and alpha-1,2,6-linked glucose in a molar ratio of 2:1:1. Three components of the PHF-associated glycolipid fraction having masses 2,416, 2,325, and 2,237 Da were observed using MALDI-MS. The least abundant, heavier mass component (2,416 Da) was best fit to a structure with a tridecamer of glucose having a single esterified C20 fatty acid (Glc13 + C20 or Glc13 + C20:1), whereas the more abundant, lower mass components were best fit to noncovalently associated glycolipid dimers, each with a glucose pentamer or hexamer having two C14, C16, or C18 esterified fatty acids {D[(Glc5 + C18) + (Glc6 + C16)] or D[(Glc5 + C14) + (Glc6 + C14)]}. The ratio of glucose to fatty acid calculated from these best-fit structures of the more abundant mass components (5.5 +/- 1.1:1.0) is in reasonable agreement with the same ratio calculated from peak integrations in the NMR spectra of acid-hydrolyzed prcPHF (6.2 +/- 1.6). Structural similarities between PHF-associated glycolipid and other glycolipid amphiphiles known to form PHF-like filaments indirectly suggest that this unique glycolipid may be an integral component of the PHF suprastructure.

Carbon-Proton Coupling Constants In The Conformational Analysis of Sugar Molecules

Publisher Summary This chapter examines the carbon–proton coupling constants involved in the conformational analysis of sugar molecules. The indirect spin–spin coupling is independent of molecular rotation. The coupling mechanism is known to involve the electron spins of the bonding electrons and is the result of a weak electron polarization. A proton signal has two 13C satellites, each having an intensity that is 0.5% of that of the corresponding total proton resonance and located symmetrically around that resonance. In addition to providing information about 1H ( 1H couplings) these satellites allow the determination of the 1JC,H couplings with good resolution. With the advent of inverse detection and the corresponding gain in sensitivity, newer schemes for the measurement of long-range coupling constants have been proposed. They are based on the heteronuclear multiple-bond correlation derivative or variants of the heteronuclear multiple-quantum coherence experiment, and also on the heteronuclear single-quantum coherence experiment. The Hamiltonian gives the total electronic kinetic energy and the magnetic interactions between electron orbital motions and nuclear magnetic moments. It is found that two-bond carbon–proton coupling constants are much smaller than one-bond couplings and their signs can be negative or positive.

Uniform 15N labeling of a fungal peptide: the structure and dynamics of an alamethicin by 15N and 1H NMR spectroscopy.

An alamethicin, secreted by the fungus Trichoderma viride and containing a glutamine at position 18 instead of the usual glutamic acid, has been uniformly labeled with 15N and purified by HPLC. The extent of 15N incorporation at individual backbone and side-chain sites was found to vary from 85% to 92%, as measured by spin-echo difference spectroscopy. The proton NMR spectrum of the peptide dissolved in methanol was assigned using correlation spectroscopies and nuclear Overhauser enhancements (NOE) measured in the rotating frame. The 15N resonances were assigned by the 2D 1H-15N correlation via heteronuclear multiple-quantum coherence experiment. NOEs and 3JNHC alpha H coupling constants strongly suggest that, in methanol, from Aib-3 to Gly-11, the peptide adopts a predominantly helical conformation, in agreement with previous 1H NMR studies [Esposito, G., Carver, J.A, Boyd, J., & Campbell, I.D. (1987) Biochemistry 26, 1043-1050; Banerjee, U., Tsui, F.-P., Balasubramanian, T.N., Marshall, G.R., & Chan, S I. (1983) J. Mol. Biol. 165, 757-775]. The conformation of the carboxyl terminus (12-20) is less well determined, partly because the amino acid composition reduces the number of NOEs and coupling constants which can be determined by 1H NMR spectroscopy. The 3JNHC alpha H in the C-terminus suggest the possibility of conformational averaging at Leu-12, Val-15, and Gln-19, an interpretation which is supported by a recent molecular dynamics simulation of the peptide [Fraternalli, F. (1990) Biopolymers 30, 1083-1099].(ABSTRACT TRUNCATED AT 250 WORDS)

A three-dimensional heteronuclear multiple-quantum coherence homonuclear hartmann-hahn NOESY experiment

Since the introduction of two-dimensional NMR a further extension into three (or more) frequency dimensions, by the insertion of one or two additional evolution times, seemed only a logical extension of the method. In the area of the high-resolution NMR, three-dimensional experiments seemed impracticable for the same reasons that were at the heart of the slow introduction of 2D NMR: very large data matrices were generated and long measuring times were needed. Increased sensitivity of NMR spectrometers, ever increasing computer power, and speed of mass storage devices have alleviated these problems. Three-dimensional NMR can nowadays be done within a reasonable amount of time and without generating excessively large data matrices as judged by today’s standards. This is borne out by a number of recently published 3D NMR experiments (1-9). The majority of these experiments entails the coupling of two 2D proton-proton correlation experiments, e.g., NOESYCOSY (3) and NOESY-HOHAHA (5). To limit the measuring time and the size of the data matrices in most cases the proton frequency domain was restricted by using selective excitation pulses, although also two 3D NOESY-HOHAHA experiments have been done in which the complete proton frequency domain was utilized (6, 7). In addition reports have appeared of 3D heteronuclear NMR experiments, where a 2D heteronucleus-proton correlation experiment is combined with a 2D protonproton correlation experiment (8, 9). For uniformly 15N- or ‘3C-enriched samples, the sensitivity of such 3D heteronuclear NMR experiments is similar to that of 3D proton NMR experiments, since the heteronucleus is detected via the attached proton and thus with proton sensitivity ( 10-12). Therefore, in terms of measuring time and size of data matrices the same conditions apply to such 3D heteronuclear NMR experiments as to 3D proton NMR. The great promise of 3D NMR experiments lies in the increased spectral resolution with respect to 2D NMR experiments, which is of prime importance in the elucidation of the molecular structure by means of NMR of proteins as well as of nucleic acids and carbohydrates. In this Communication we present a new 3D NMR experiment in which a ‘Hdetected heteronuclear multiple-quantum coherence experiment (HMQC) ( 10-12)