Sensitivity improvement and new acquisition scheme of heteronuclear active-coupling-pattern-tilting spectroscopy

A simplified phase-cycling scheme for heteronuclear active-coupling-pattern tilting (ACT) spectroscopy is presented. It is demonstrated that the theoretically expected twofold sensitivity gain over earlier implementations can be experimentally realized. A further intensity increase by a factor of 2 is obtained with standard sensitivity-enhancement pulse-sequence elements. The HSQC-HECADE sequence presented is designed for an accurate determination of heteronuclear one-bond and, with subsequent I-spin isotropic mixing, long-range coupling constants. As an exemplary application, the determination of the (3)J(N,Hbeta) coupling constants in a peptide at natural isotope abundance is demonstrated. Additionally, a new polarization-transfer step for the long-range HSQC-HECADE experiment is proposed which avoids a fixed delay tuned to a specific coupling-constant value. Thus, the long-range correlation experiment does not require prior knowledge of the coupling constants to be measured and yields more uniform cross-peak intensity for a broad range of active coupling constants.     

High-resolution heteronuclear correlation spectroscopy in solid state NMR of aligned samples

A new two-dimensional scheme is proposed for accurate measurements of high-resolution chemical shifts and heteronuclear dipolar couplings in NMR of aligned samples. Both the (1)H chemical shifts and the (1)H-(15)N dipolar couplings are evolved in the indirect dimension while the (15)N chemical shifts are detected. This heteronuclear correlation (HETCOR) spectroscopy yields high-resolution (1)H chemical shifts split by the (1)H-(15)N dipolar couplings in the indirect dimension and the (15)N chemical shifts in the observed dimension. The advantages of the HETCOR technique are illustrated for a static (15)N-acetyl-valine crystal sample and a (15)N-labeled helical peptide sample aligned in hydrated lipid bilayers.     

Proton-detected heteronuclear single quantum correlation NMR spectroscopy in rigid solids with ultra-fast MAS

In this article, we show the potential for utilizing proton-detected heteronuclear single quantum correlation (HSQC) NMR in rigid solids under ultra-fast magic angle spinning (MAS) conditions. The indirect detection of carbon-13 from coupled neighboring hydrogen nuclei provides a sensitivity enhancement of 3- to 4-fold in crystalline amino acids over direct-detected versions. Furthermore, the sensitivity enhancement is shown to be significantly larger for disordered solids that display inhomogeneously broadened carbon-13 spectra. Latrodectus hesperus (Black Widow) dragline silk is given as an example where the sample is mass-limited and the sensitivity enhancement for the proton-detected experiment is 8- to 13-fold. The ultra-fast MAS proton-detected HSQC solid-state NMR technique has the added advantage that no proton homonuclear decoupling is applied during the experiment. Further, well-resolved, indirectly observed carbon-13 spectra can be obtained in some cases without heteronuclear proton decoupling.     

Indirectly detected heteronuclear correlation solid-state NMR spectroscopy of naturally abundant 15N nuclei

Two-dimensional indirectly detected through-space and through-bond ¹H{¹⁵N} solid-state NMR experiments utilizing fast magic angle spinning (MAS) and homonuclear multipulse ¹H decoupling are evaluated. Remarkable efficiency of polarization transfer can be achieved at a MAS rate of 40 kHz by both cross-polarization and INEPT, which makes these methods applicable for routine characterizations of natural abundance solids. The first measurement of 2D ¹H{¹⁵N} HETCOR spectrum of natural abundance surface species is also reported.     

High resolution heteronuclear correlation NMR spectroscopy between quadrupolar nuclei and protons in the solid state

A high resolution two-dimensional solid state NMR experiment is presented that correlates half-integer quadrupolar spins with protons. In this experiment the quadrupolar nuclei evolve during t1 under a split-t1, FAM-enhanced MQMAS pulse scheme. After each t1 period ending at the MQMAS echo position, single quantum magnetization is transferred, via a cross polarization process in the mixing time, from the quadrupolar nuclei to the protons. High-resolution proton signals are then detected in the t2 time domain during wPMLG5* homonuclear decoupling. The experiment has been demonstrated on a powder sample of sodium citrate and 23Na-1H 2D correlation spectra have been obtained. From the HETCOR spectra and the regular MQMAS spectrum, the three crystallographically inequivalent Na+ sites in the asymmetric unit were assigned. This MQMAS-wPMLG HETCOR pulse sequence can be used for spectral editing of half-integer quadrupolar nuclei coupled to protons.     

Through-bond heteronuclear single-quantum correlation spectroscopy in solid-state NMR, and comparison to other through-bond and through-space experiments

A new through-bond carbon-proton correlation technique, the MAS-J-HSQC experiment, is described for solid-state NMR. This new pulse scheme is compared experimentally with the previously proposed MAS-J-HMQC experiment in terms of proton resolution on a model sample of powdered L-alanine. We show that for natural abundance compounds, the MAS-J-HMQC and MAS-J-HSQC experiments give about the same proton resolution, whereas, for (13)C-labeled materials, narrower proton linewidths are obtained with the MAS-J-HSQC experiment. In addition we show that in scalar as well as in dipolar heteronuclear shift correlation experiments, when the proton chemical shift is encoded by the evolution of a single-quantum coherence, the proton resolution can be enhanced by simply adding a 180 degrees carbon pulse in the middle of the t(1) evolution time.     

Double-quantum filtered heteronuclear correlation spectroscopy under magic angle spinning

We present a simple experimental method to extract the van Vleck second moment of a multiple-spin system under high-resolution condition. The idea is to incorporate a double-quantum (DQ) filter into the pulse sequence of heteronuclear correlation spectroscopy so that a DQ excitation profile can be obtained by measuring a series of 2D spectra. The effects of spinning frequency and proton decoupling are demonstrated on the measurements of two model compounds, viz. hydroxyapatite and brushite. Based on the results obtained for the model compounds, the P-31 homonuclear second moment of the apatite component in rat dentin is characterized. The method is generally suited for the study of bone, enamel and dentin.     

Three-dimensional electrophoretic NMR correlation spectroscopy

A novel method of three-dimensional electrophoretic NMR correlation spectroscopy (3D EP-COSY) has been proposed, developed, and implemented. It has a demonstrated potential of facilitating simultaneous structural assignments of multiple proteins in mixtures. The principle is to add a pulsed DC electric field that introduces a new dimension of electrophoretic flow, in which resonances of different molecules can be separated by their electrophoretic migration rates without physical separation. As a result, two COSY spectra were simultaneously obtained in a single 3D EP-COSY experiment from a mixture of 150 mM l-aspartic acid and 148 mM 4, 9-dioxa-1,12-dodecanediamine with concurrent resolution of their chemical shifts and J-coupling constants. This approach creates a new horizon of multidimensional electrophoretic NMR. The technical advance opens doors for structure characterization of complex protein systems and protein interactions, which are at the basis of biochemical mechanisms and the phenomena of living systems.     

Gradient-enhanced heteronuclear correlation spectroscopy: theory and experimental aspects. 1992

A detailed theoretical and experimental treatment is given for gradient-enhanced heteronuclear correlation spectroscopy. Both multiple-quantum and single-quantum sequences are described. In addition to a comparison with conventional experiments using phase cycling, the effects of different gradient combinations are examined with respect to artifacts occurring in the heteronuclear dimension. The influence of gradient performance and diffusion on sensitivity is discussed. Approaches to attain phase-sensitive spectra are also analyzed.     

H-start for exclusively heteronuclear NMR spectroscopy: The case of intrinsically disordered proteins

Here, we present a series of exclusively heteronuclear multidimensional NMR experiments, based on ¹³C direct detection, which exploit the ¹H polarization as a starting source to increase the signal-to-noise ratio. This contributes to make this spectroscopy more useful and usable. Examples are reported for a suitable system such as securin, an intrinsically disordered protein of 22 kDa.     

High-resolution heteronuclear correlation spectroscopy based on spatial encoding and coherence transfer in inhomogeneous fields

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy has been proven to be a powerful technique for chemical, biological, and medical studies. Heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) are two frequently used 2D NMR methods. In combination with spatially encoded techniques, a heteronuclear 2D NMR spectrum can be acquired in several seconds and may be applied to monitoring chemical reactions. However, it is difficult to obtain high-resolution NMR spectra in inhomogeneous fields. Inspired by the idea of tracing the difference of precession frequencies between two different spins to yield high-resolution spectra, we propose a method with correlation acquisition option and J-resolved-like acquisition option to ultrafast obtain high-resolution HSQC/HMBC spectra and heteronuclear J-resolved-like spectra in inhomogeneous fields.     

1H assisted 13C/15N heteronuclear correlation spectroscopy in oriented sample solid-state NMR of single crystal and magnetically aligned samples

(1)H-irradiation under mismatched Hartmann-Hahn conditions provides an alternative mechanism for carrying out (15)N/(13)C transfers in triple-resonance heteronuclear correlation spectroscopy (HETCOR) on stationary samples of single crystals and aligned samples of biopolymers, which improve the efficiency especially when the direct (15)N-(13)C dipolar couplings are small. In many cases, the sensitivity is improved by taking advantage of the (13)C(α) labeled sites in peptides and proteins with (13)C detection. The similarities between experimental and simulated spectra demonstrate the validity of the recoupling mechanism and identify the potential for applying these experiments to virus particles or membrane proteins in phospholipid bilayers; however, further development is needed in order to derive quantitative distance and angular constraints from these measurements.     

Characteristics of zero-quantum correlation spectroscopy in MAS NMR experiments

Zero-quantum coherence generation and reconversion in magic-angle spinning solid-state NMR is analyzed. Two methods are discussed based on implementations using symmetry-based pulse sequences that utilize either isotropic J couplings or dipolar couplings. In either case, the decoupling of abundant proton spins plays a crucial role for the efficiency of the zero-quantum generation. We present optimized sequences for measuring zero-quantum single-quantum correlation spectra in solids, achieving an efficiency of 50% in ubiquitin. The advantages and disadvantages of zero-quantum single-quantum over single-quantum single-quantum correlation spectroscopy are explored, and similarities and differences with double-quantum single-quantum correlation spectroscopy are discussed. Finally, possible application of zero-quantum single-quantum experiments to polypeptides, where it can lead to better spectral resolution is investigated using ubiquitin, where we find high efficiency and high selectivity, but also increased line widths in the MQ dimension.     

Sensitivity and resolution enhancement in solid-state NMR spectroscopy of bicelles

Magnetically aligned bicelles are becoming attractive model membranes to investigate the structure, dynamics, geometry, and interaction of membrane-associated peptides and proteins using solution- and solid-state NMR experiments. Recent studies have shown that bicelles are more suitable than mechanically aligned bilayers for multidimensional solid-state NMR experiments. In this work, we describe experimental aspects of the natural abundance (13)C and (14)N NMR spectroscopy of DMPC/DHPC bicelles. In particular, approaches to enhance the sensitivity and resolution and to quantify radio-frequency heating effects are presented. Sensitivity of (13)C detection using single pulse excitation, conventional cross-polarization (CP), ramp-CP, and NOE techniques are compared. Our results suggest that the proton decoupling efficiency of the FLOPSY pulse sequence is better than that of continuous wave decoupling, TPPM, SPINAL, and WALTZ sequences. A simple method of monitoring the water proton chemical shift is demonstrated for the measurement of sample temperature and calibration of the radio-frequency-induced heating in the sample. The possibility of using (14)N experiments on bicelles is also discussed.     

Probing membrane topology by high-resolution 1H-13C heteronuclear dipolar solid-state NMR spectroscopy

Membrane topology changes introduced by the association of biologically pertinent molecules with membranes were analyzed utilizing the (1)H-(13)C heteronuclear dipolar solid-state NMR spectroscopy technique (SAMMY) on magnetically aligned phospholipid bilayers (bicelles). The phospholipids (1)H-(13)C dipolar coupling profiles lipid motions at the headgroup, glycerol backbone, and the acyl chain region. The transmembrane segment of phospholamban, the antimicrobial peptide (KIGAKI)(3) and cholesterol were incorporated into the bicelles, respectively. The lipids (1)H-(13)C dipolar coupling profiles exhibit different shifts in the dipolar coupling contour positions upon the addition of these molecules, demonstrating a variety of interaction mechanisms exist between the biological molecules and the membranes. The membrane topology changes revealed by the SAMMY pulse sequence provide a complete screening method for analyzing how these biologically active molecules interact with the membrane.