Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

Provided that ¹³C‐detected NMR experiments are either preferable or complementary to ¹H detection, we report here tools to determine C^α? C′, C′? N, and C^α? H^α residual dipolar couplings on the basis of the CON experiment. The coupling constants determined on ubiquitin are consistent with the subset measured with the ¹H‐detected HNCO sequences. Since the utilization of residual dipolar couplings may depend on the mobility of the involved nuclei, we also provide tools to measure longitudinal and transverse relaxation rates of N and C′. This new set of experiments is a further development of a whole strategy based on ¹³C direct‐detection NMR spectroscopy for the study of biological macromolecules.     

Resolution Enhancement in SEA XLOC for Heteronuclear NMR Long-Range Correlation

It is shown how the resolution in SEA XLOC NMR spectra for distinguishing between heteronuclear two- and three-bond correlations for all ¹³C multiplicities can be improved by a modified experiment delivering absorptive profiles in the indirect dimension. The method is demonstrated with applications to ibuprofen and strychnine.     

A definitive NMR solution for a simple and accurate measurement of the magnitude and the sign of small heteronuclear coupling constants on protonated and non-protonated carbon atoms

Simply successful: a proton-selective HSQMBC-TOCSY experiment can be used to measure small proton-carbon ((n)J(CH); n>1) coupling constants on both protonated and non-protonated carbon atoms. The method combines in a single pulse scheme all the benefits of the widely used HSQMBC and HSQC-TOCSY experiments. The magnitude and the sign of (n)J(CH) can be determined simply with excellent accuracy.     

Precise Measurement of Long‐Range Heteronuclear Coupling Constants by a Novel Broadband Proton–Proton‐Decoupled CPMG‐HSQMBC Method

A broadband proton–proton‐decoupled CPMG‐HSQMBC method for the precise and direct measurement of long‐range heteronuclear coupling constants is presented. The Zangger–Sterk‐based homodecoupling scheme reported herein efficiently removes unwanted proton–proton splittings from the heteronuclear multiplets, so that the desired heteronuclear couplings can be determined simply by measuring frequency differences between singlet maxima in the resulting spectra. The proposed pseudo‐1D/2D pulse sequences were tested on nucleotides, a metal complex incorporating P heterocycles, and diglycosyl (di)selenides, as well as on other carbohydrate derivatives, for the extraction of ⁿJ(¹H,³¹P), ⁿJ(¹H,⁷⁷Se), and ⁿJ(¹H,¹³C) values, respectively.     

Pure In‐Phase Heteronuclear Correlation NMR Experiments

A general NMR approach to provide pure in‐phase (PIP) multiplets in heteronuclear correlation experiments is described. The implementation of a zero‐quantum filter efficiently suppresses any unwanted anti‐phase contributions that usually distort the multiplet pattern of cross‐peaks and can hamper their analysis. The clean pattern obtained in PIP‐HSQMBC experiments is suitable for a direct extraction of coupling constants in resolved signals, for a peak‐fitting process from a reference signal, and for the application of the IPAP technique in non‐resolved multiplets.     

1D NMR Homodecoupled 1H Spectra with Scalar Coupling Constants from 2D NemoZS‐DIAG Experiments

A two‐dimensional liquid‐state NMR experiment cleanly separating chemical shifts and scalar couplings information is introduced. This DIAG experiment takes advantage of a drastic reduction of the spectral window in the indirect dimension to be quickly recorded and of a new non‐equidistant modulation of the selective pulse to improve the sensitivity of the broadband homodecoupling Zangger–Sterk sequence element by one order of magnitude. A simple automatic analysis results in 1D spectra displaying singlets and lists of the scalar couplings for first‐order multiplets. This facilitates the analysis of 1D spectra by resolving multiplets based on their differences in chemical shifts and coupling structures.     

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 ⁿJ(¹H,⁷⁷Se) and ⁿJ(¹H,¹³C) values, respectively, in carbohydrates; further extensions can be envisioned in any J‐based structural and conformational studies.     

Scalar Coupling Constants—Their Analysis and Their Application for the Elucidation of Structures

Since their discovery in the early fifties, scalar/coupling constants have been of great interest to the NMR spectroscopist. Their impact on structure determination by NMR spectroscopy is founded on the fact that the size of the coupling constant is directly related to molecular conformation. Today, for most chemical substances the parameters for the Karplus relationship, which relates the vicinial (3-bond) coupling constant to the dihedral angle, have been determined. In addition to proton–proton distances, the application of coupling constants in modern conformational analysis is indispensable. In the study of larger molecules which are of current interest, more and more involved experiments are necessary in order to overcome signal overlap and increasing line widths. A large number of experimental techniques for the determination of coupling constants has been developed; however, for this reason the choice of the most appropriate experiment to use has become more difficult. This decision must be made carefully to maximize instrument usage and obtain the largest number of couplings with the greatest accuracy possible. Many of the computer programs used in structure calculations can directly apply coupling constant restraints, similar to proton–proton distances developed from NOEs. Therefore, not only is the quality of the structure improved, but the molecular motions (internal dynamics) are better described. In this article, we review the techniques that exist today with particular attention paid to helping the non-expert to choose the appropriate experiment for the problem at hand. In addition, the use of coupling constants in computer simulations are discussed.     

113Cd NMR Experiments Reveal an Unusual Metal Cluster in the Solution Structure of the Yeast Splicing Protein Bud31p

Establishing the binding topology of structural zinc ions in proteins is an essential part of their structure determination by NMR spectroscopy. Using ¹¹³Cd NMR experiments with ¹¹³Cd‐substituted samples is a useful approach but has previously been limited mainly to very small protein domains. Here we used ¹¹³Cd NMR spectroscopy during structure determination of Bud31p, a 157‐residue yeast protein containing an unusual Zn₃Cys₉ cluster, demonstrating that recent hardware developments make this approach feasible for significantly larger systems.     

Decoupling two-dimensional NMR spectroscopy in both dimensions: pure shift NOESY and COSY

An increase in the resolving power in 2D NMR spectra is obtained by collapsing 2D signals with multiplet structure into 2D singlets. This resolution gain is achieved by combining 2D experiments with pure shift techniques and covariance processing (see picture). The method should be of value in both manual and automated structure determination.     

Computational NMR coupling constants: Shifting and scaling factors for evaluating 1JCH

Optimized shifting and/or scaling factors for calculating one‐bond carbon–hydrogen spin–spin coupling constants have been determined for 35 combinations of representative functionals (PBE, B3LYP, B3P86, B97‐2 and M06‐L) and basis sets (TZVP, HIII‐su3, EPR‐III, aug‐cc‐pVTZ‐J, ccJ‐pVDZ, ccJ‐pVTZ, ccJ‐pVQZ, pcJ‐2 and pcJ‐3) using 68 organic molecular systems with 88 ¹J_CH couplings including different types of hybridized carbon atoms. Density functional theory assessment for the determination of ¹J_CH coupling constants is examined, comparing the computed and experimental values. The use of shifting constants for obtaining the calculated coupling improves substantially the results, and most models become qualitatively similar. Thus, for the whole set of couplings and for all approaches excluding those using the M06 functional, the root‐mean‐square deviations lie between 4.7 and 16.4 Hz and are reduced to 4–6.5 Hz when shifting constants are considered. Alternatively, when a specific rovibrational contribution of 5 Hz is subtracted from the experimental values, good results are obtained with PBE, B3P86 and B97‐2 functionals in combination with HIII‐su3, aug‐cc‐pVTZ‐J and pcJ‐2 basis sets. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous definition of high resolution protein structure and backbone conformational dynamics using NMR residual dipolar couplings.

Despite the importance of molecular dynamics for biological activity, most approaches to protein structure determination, whether based on crystallographic or solution studies, propose three-dimensional atomic representations of a single configuration that take no account of conformational fluctuation. Non-averaged anisotropic NMR interactions, such as residual dipolar couplings, that become measurable under conditions of weak alignment, provide sensitive probes of both molecular structure and dynamics. Residual dipolar couplings are becoming increasingly powerful for the study of proteins in solution. In this minireview we present their use for the simultaneous determination of protein structure and dynamics.     

Giant Residual Dipolar 13C–1H Couplings in High‐Spin Organoiron Complexes: Elucidation of Their Structures in Solution by 13C NMR Spectroscopy

High‐spin Fe^II–alkyl complexes with bis(pyridylimino)isoindolato ligands were synthesized and their paramagnetic ¹H and ¹³C NMR spectra were analyzed comprehensively. The experimental ¹³C—¹H coupling values are temperature (T^?1)‐ as well as magnetic‐field (B²)‐dependent and deviate considerably from typical scalar ¹J_CH couplings constants. This deviation is attributed to residual dipolar couplings (RDCs), which arise from partial alignment of the complexes in the presence of a strong magnetic field. The analysis of the experimental RDCs allows an unambiguous assignment of all ¹³C NMR resonances and, additionally, a structural refinement of the conformation of the complexes in solution. Moreover the RDCs can be used for the analysis of the alignment tensor and hence the tensor of the anisotropy of the magnetic susceptibility.     

Heteronuclear NMR Spectroscopy as a Surface‐Selective Technique: A Unique Look at the Hydroxyl Groups of γ‐Alumina.

The surface hydroxyl groups of γ‐alumina dehydroxylated at 500 °C were studied by a combination of one‐ and two‐dimensional homo‐ and heteronuclear ¹H and ²⁷Al NMR spectroscopy at high magnetic field. In particular, by harnessing ¹H–²⁷Al dipolar interactions, a high selectivity was achieved in unveiling the topology of the alumina surface. The terminal versus bridging character of the hydroxyl groups observed in the ¹H magic‐angle spinning (MAS) NMR spectrum was demonstrated thanks to ¹H–²⁷Al RESPDOR (resonance‐echo saturation‐pulse double‐resonance). In a further step the hydroxyl groups were assigned to their aluminium neighbours thanks to a {¹H}‐²⁷Al dipolar heteronuclear multiple quantum correlation (D‐HMQC), which was used to establish a first coordination map. Then, in combination with ¹H–¹H double quantum (DQ) MAS, these elements helped to reveal intimate structural features of the surface hydroxyls. Finally, the nature of a peculiar reactive hydroxyl group was demonstrated following this methodology in the case of CO₂ reactivity with alumina.     

Extending the scope of NMR spectroscopy with microcoil probes

Capillary NMR (CapNMR) spectroscopy has emerged as a major breakthrough for increasing the mass-sensitivity of NMR spectroscopic analysis and enabling the combination of NMR spectroscopy with other analytical techniques. Not only is the acquisition of high-sensitivity spectra getting easier but the quality of CapNMR spectra obtained in many small-molecule applications exceeds what can be accomplished with conventional designs. This Minireview discusses current CapNMR technology and its applications for the characterization of mass-limited, small-molecule and protein samples, the rapid screening of small-molecule or protein libraries, as well as hyphenated techniques that combine CapNMR with other analytical methods.