Chemical Shift Correlation Spectra Research Articles

Using Abundant 1H Polarization to Enhance the Sensitivity of Solid-State NMR Spectroscopy.

Solid-state NMR spectroscopy has been playing a significant role in elucidating the structures and dynamics of materials and proteins at the atomic level for decades. As an extremely abundant nucleus with a very high gyromagnetic ratio, protons are widely present in most organic/inorganic materials. Thus, this Perspective highlights the advantages of proton detection at fast magic-angle spinning (MAS) and presents strategies to utilize and exhaust 1H polarization to achieve signal sensitivity enhancement of solid-state NMR spectroscopy, enabling substantial time savings and extraction of more structural and dynamics information per unit time. Those strategies include developing sensitivity-enhanced single-channel 1H multidimensional NMR spectroscopy, implementing multiple polarization transfer steps in each scan to enhance low-γ nuclei signals, and making full use of 1H polarization to obtain homonuclear and heteronuclear chemical shift correlation spectra in a single experiment. Finally, outlooks and perspectives are provided regarding the challenges and future for the further development of sensitivity-enhanced proton-based solid-state NMR spectroscopy.

Rapid Structural Analysis of Minute Quantities of Organic Solids by Exhausting 1H Polarization in Solid-State NMR Spectroscopy Under Fast Magic Angle Spinning.

Solid-state nuclear magnetic resonance (NMR) often suffers from significant limitations due to the inherent low signal sensitivity when low-γ nuclei are involved. Herein, we report an elegant solid-state NMR approach for rapid structural analysis of minute amounts of organic solids. By encoding staggered chemical shift evolution in the indirect dimension and staggered acquisition in the 1H dimension, a proton-detected homonuclear 1H/1H and heteronuclear 13C/1H chemical shift correlation (HETCOR) spectrum can be obtained simultaneously in a single experiment at a fast magic-angle-spinning (MAS) condition with barely increasing the experimental time. We further show that during the conventional 1H-detected HETCOR experimental time, multiple homonuclear 1H/1H correlation spectra can be recorded in addition to the HETCOR spectrum, enabling the determination of 1H-1H distances. We establish that abundant 1H polarization can be efficiently manipulated and fully utilized in proton-detected solid-state NMR spectroscopy for extraction of more critical structural information and thus reduction of the total experimental time.

Exploiting heterogeneous time scale of dynamics to enhance 2D HETCOR solid-state NMR sensitivity.

Multidimensional solid-state NMR spectroscopy plays a significant role in offering atomic-level insights into molecular systems. In particular, heteronuclear chemical shift correlation (HETCOR) experiments could provide local chemical and structural information in terms of spatial heteronuclear proximity and through-bond connectivity. In solid state, the transfer of magnetization between heteronuclei, a key step in HETCOR experiments, is usually achieved using cross-polarization (CP) or insensitive nuclei enhanced by polarization transfer (INEPT) depending on the sample characteristics and magic-angle-spinning (MAS) frequency. But, for a multiphase system constituting molecular components that differ in their time scales of mobilities, CP efficiency is pretty low for mobile components because of the averaging of heteronuclear dipolar couplings whereas INEPT is inefficient for immobile components due to the short T2 and can yield through-space connectivity due to strong proton spin diffusion for immobile components especially under moderate spinning speeds. Herein, in this study we present two 2D pulse sequences that enable the sequential acquisition of 13C/1H HETCOR NMR spectra for the rigid and mobile components by taking full advantage of the abundant proton magnetization in a single experiment with barely increasing the overall experimental time. In particular, the 13C-detected HETCOR experiment could be applied under slow MAS conditions, where a multiple-pulse sequence is typically employed to enhance 1H spectral resolution in the indirect dimension. In contrast, the 1H-detected HETCOR experiment should be applied under ultrafast MAS, where CP and heteronuclear nuclear Overhauser effect (NOE) polarization transfer are combined to enhance 13C signal intensities for mobile components. These pulse sequences are experimentally demonstrated on two model systems to obtain 2D 13C/1H chemical shift correlation spectra of rigid and mobile components independently and separately. These pulse sequences can be used for dynamics based spectral editing and resonance assignments. Therefore, we believe the proposed 2D HETCOR NMR pulse sequences will be beneficial for the structural studies of heterogeneous systems containing molecular components that differ in their time scale of motions for understanding the interplay of structures and properties.

Spin-echo based diagonal peak suppression in solid-state MAS NMR homonuclear chemical shift correlation spectra

The feasibility of using the spin-echo based diagonal peak suppression method in solid-state MAS NMR homonuclear chemical shift correlation experiments is demonstrated. A complete phase cycling is designed in such a way that in the indirect dimension only the spin diffused signals are evolved, while all signals not involved in polarization transfer are refocused for cancellation. A data processing procedure is further introduced to reconstruct this acquired spectrum into a conventional two-dimensional homonuclear chemical shift correlation spectrum. A uniformly 13C, 15N labeled Fmoc-valine sample and the transmembrane domain of a human protein, LR11 (sorLA), in native Escherichia coli membranes have been used to illustrate the capability of the proposed method in comparison with standard 13C–13C chemical shift correlation experiments.

Computer-aided Dereplication and Structure Elucidation of Natural Products at the University of Reims.

Natural product chemistry began in Reims, France, in a pharmacognosy research laboratory whose main emphasis was the isolation and identification of bioactive molecules, following the guidelines of chemotaxonomy. The structure elucidation of new compounds of steadily increasing complexity favored the emergence of methodological work in nuclear magnetic resonance. As a result, our group was the first to report the use of proton-detected heteronuclear chemical shift correlation spectra for the computer-assisted structure elucidation of small organic molecules driven by atom proximity relationships and without relying on databases. The early detection of known compounds appeared as a necessity in order to deal more efficiently with complex plant extracts. This goal was reached by an original combination of mixture fractionation by centrifugal partition chromatography, analysis by 13 C NMR, digital data reduction and alignment, hierarchical data clustering, and computer database search.

Constant-time 2D and 3D through-bond correlation NMR spectroscopy of solids under 60 kHz MAS.

Establishing connectivity and proximity of nuclei is an important step in elucidating the structure and dynamics of molecules in solids using magic angle spinning (MAS) NMR spectroscopy. Although recent studies have successfully demonstrated the feasibility of proton-detected multidimensional solid-state NMR experiments under ultrafast-MAS frequencies and obtaining high-resolution spectral lines of protons, assignment of proton resonances is a major challenge. In this study, we first re-visit and demonstrate the feasibility of 2D constant-time uniform-sign cross-peak correlation (CTUC-COSY) NMR experiment on rigid solids under ultrafast-MAS conditions, where the sensitivity of the experiment is enhanced by the reduced spin-spin relaxation rate and the use of low radio-frequency power for heteronuclear decoupling during the evolution intervals of the pulse sequence. In addition, we experimentally demonstrate the performance of a proton-detected pulse sequence to obtain a 3D (1)H/(13)C/(1)H chemical shift correlation spectrum by incorporating an additional cross-polarization period in the CTUC-COSY pulse sequence to enable proton chemical shift evolution and proton detection in the incrementable t1 and t3 periods, respectively. In addition to through-space and through-bond (13)C/(1)H and (13)C/(13)C chemical shift correlations, the 3D (1)H/(13)C/(1)H experiment also provides a COSY-type (1)H/(1)H chemical shift correlation spectrum, where only the chemical shifts of those protons, which are bonded to two neighboring carbons, are correlated. By extracting 2D F1/F3 slices ((1)H/(1)H chemical shift correlation spectrum) at different (13)C chemical shift frequencies from the 3D (1)H/(13)C/(1)H spectrum, resonances of proton atoms located close to a specific carbon atom can be identified. Overall, the through-bond and through-space homonuclear/heteronuclear proximities determined from the 3D (1)H/(13)C/(1)H experiment would be useful to study the structure and dynamics of a variety of chemical and biological solids.

Proton-detected 3D (1)H/(13)C/(1)H correlation experiment for structural analysis in rigid solids under ultrafast-MAS above 60 kHz.

A proton-detected 3D (1)H/(13)C/(1)H chemical shift correlation experiment is proposed for the assignment of chemical shift resonances, identification of (13)C-(1)H connectivities, and proximities of (13)C-(1)H and (1)H-(1)H nuclei under ultrafast magic-angle-spinning (ultrafast-MAS) conditions. Ultrafast-MAS is used to suppress all anisotropic interactions including (1)H-(1)H dipolar couplings, while the finite-pulse radio frequency driven dipolar recoupling (fp-RFDR) pulse sequence is used to recouple dipolar couplings among protons and the insensitive nuclei enhanced by polarization transfer technique is used to transfer magnetization between heteronuclear spins. The 3D experiment eliminates signals from non-carbon-bonded protons and non-proton-bonded carbons to enhance spectral resolution. The 2D (F1/F3) (1)H/(1)H and 2D (13)C/(1)H (F2/F3) chemical shift correlation spectra extracted from the 3D spectrum enable the identification of (1)H-(1)H proximity and (13)C-(1)H connectivity. In addition, the 2D (F1/F2) (1)H/(13)C chemical shift correlation spectrum, incorporated with proton magnetization exchange via the fp-RFDR recoupling of (1)H-(1)H dipolar couplings, enables the measurement of proximities between (13)C and even the remote non-carbon-bonded protons. The 3D experiment also gives three-spin proximities of (1)H-(1)H-(13)C chains. Experimental results obtained from powder samples of L-alanine and L-histidine ⋅ H2O ⋅ HCl demonstrate the efficiency of the 3D experiment.

HN-NCA heteronuclear TOCSY-NH experiment for (1)H(N) and (15)N sequential correlations in ((13)C, (15)N) labelled intrinsically disordered proteins.

A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue 'i' with that of residues 'i-1' and 'i+1' in ((13)C, (15)N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploits the favourable relaxation properties of IDPs and the presence of (1) J CαN and (2) J CαN couplings to transfer the (15)N x magnetisation from amino acid residue 'i' to adjacent residues via the application of a band-selective (15)N-(13)C(α) heteronuclear cross-polarisation sequence of ~100ms duration. Employing non-uniform sampling in the indirect dimensions, the efficacy of the approach has been demonstrated by the acquisition of 3D HNN chemical shift correlation spectra of α-synuclein. The experimental performance of the RF pulse sequence has been compared with that of the conventional INEPT-based HN(CA)NH pulse scheme. As the availability of data from both the HCCNH and HNN experiments will make it possible to use the information extracted from one experiment to simplify the analysis of the data of the other and lead to a robust approach for unambiguous backbone and side-chain resonance assignments, a time-saving strategy for the simultaneous collection of HCCNH and HNN data is also described.

Proton-Detected Solid-State NMR Spectroscopy of Bone with Ultrafast Magic Angle Spinning.

While obtaining high-resolution structural details from bone is highly important to better understand its mechanical strength and the effects of aging and disease on bone ultrastructure, it has been a major challenge to do so with existing biophysical techniques. Though solid-state NMR spectroscopy has the potential to reveal the structural details of bone, it suffers from poor spectral resolution and sensitivity. Nonetheless, recent developments in magic angle spinning (MAS) NMR technology have made it possible to spin solid samples up to 110 kHz frequency. With such remarkable capabilities, 1H-detected NMR experiments that have traditionally been challenging on rigid solids can now be implemented. Here, we report the first application of multidimensional 1H-detected NMR measurements on bone under ultrafast MAS conditions to provide atomistic-level elucidation of the complex heterogeneous structure of bone. Our investigations demonstrate that two-dimensional 1H/1H chemical shift correlation spectra for bone are obtainable using fp-RFDR (finite-pulse radio-frequency-driven dipolar recoupling) pulse sequence under ultrafast MAS. Our results infer that water exhibits distinct 1H−1H dipolar coupling networks with the backbone and side-chain regions in collagen. These results show the promising potential of proton-detected ultrafast MAS NMR for monitoring structural and dynamic changes caused by mechanical loading and disease in bone.

On the problem of resonance assignments in solid state NMR of uniformly 15N, 13C-labeled proteins

Determination of accurate resonance assignments from multidimensional chemical shift correlation spectra is one of the major problems in biomolecular solid state NMR, particularly for relative large proteins with less-than-ideal NMR linewidths. This article investigates the difficulty of resonance assignment, using a computational Monte Carlo/simulated annealing (MCSA) algorithm to search for assignments from artificial three-dimensional spectra that are constructed from the reported isotropic 15N and 13C chemical shifts of two proteins whose structures have been determined by solution NMR methods. The results demonstrate how assignment simulations can provide new insights into factors that affect the assignment process, which can then help guide the design of experimental strategies. Specifically, simulations are performed for the catalytic domain of SrtC (147 residues, primarily β-sheet secondary structure) and the N-terminal domain of MLKL (166 residues, primarily α-helical secondary structure). Assuming unambiguous residue-type assignments and four ideal three-dimensional data sets (NCACX, NCOCX, CONCA, and CANCA), uncertainties in chemical shifts must be less than 0.4ppm for assignments for SrtC to be unique, and less than 0.2ppm for MLKL. Eliminating CANCA data has no significant effect, but additionally eliminating CONCA data leads to more stringent requirements for chemical shift precision. Introducing moderate ambiguities in residue-type assignments does not have a significant effect.

MAS solid state NMR of proteins: simultaneous ¹⁵N- ¹³CA and ¹⁵N- ¹³CO dipolar recoupling via low-power symmetry-based RF pulse schemes.

The generation of efficient RN n (ν)s,(ν)k symmetry-based low-power RF pulse schemes for simultaneous (15)N-(13)CA and (15)N-(13)CO dipolar recoupling is demonstrated. The method involves mixing schemes employing phase and amplitude-modulated dual band-selective 180° pulses as basic "R" element and tailoring of the RF field-modulation profile of the 180° pulses so as to obtain efficient magnetisation transfer characteristics over the resonance offset range of the nuclei involved. Mixing schemes leading to simultaneous (15)N-(13)CA and (15)N-(13)CO dipolar recoupling would permit the one-shot acquisition of different chemical shift correlation spectra that are typically utilized for protein backbone resonance assignments and thereby save data acquisition time. At representative MAS frequencies the efficacies of the mixing schemes presented here have been experimentally demonstrated via the simultaneous acquisition of {3D CONH and 3D CANH}, {3D CONH and 3D CO(CA)NH} and {3D CONH, 3D CANH, 3D CO(CA)NH and 3D CA(CO)NH} spectra generated via the magnetisation transfer pathways (1)H → (13)CO → (15)N → (1)H (CONH), (1)H → (13)CA → (15)N → (1)H (CANH) and (1)H → (13)CO → (13)CA → (15)N → (1)H (CO(CA)NH) and (1)H → (13)CA → (13)CO → (15)N → (1)H (CA(CO)NH).

An approach to sequential NMR assignments of proteins: application to chemical shift restraint-based structure prediction.

A procedure for the simultaneous acquisition of {HNCOCANH & HCCCONH} chemical shift correlation spectra employing sequential [Formula: see text] data acquisition for moderately sized proteins is presented. The suitability of the approach for obtaining sequential resonance assignments, including complete [Formula: see text] and [Formula: see text] chemical shift information, is demonstrated experimentally for a [Formula: see text] and [Formula: see text] labelled sample of the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus. The chemical shift information obtained was used to calculate the global fold of this winged helix domain via CS-Rosetta. This demonstrates that our procedure provides a reliable and straight-forward protocol for a quick global fold determination of moderately-sized proteins.

Sequential acquisition of multi-dimensional heteronuclear chemical shift correlation spectra with 1H detection

RF pulse schemes for the simultaneous acquisition of heteronuclear multi-dimensional chemical shift correlation spectra, such as {HA(CA)NH & HA(CACO)NH}, {HA(CA)NH & H(N)CAHA} and {H(N)CAHA & H(CC)NH}, that are commonly employed in the study of moderately-sized protein molecules, have been implemented using dual sequential 1H acquisitions in the direct dimension. Such an approach is not only beneficial in terms of the reduction of experimental time as compared to data collection via two separate experiments but also facilitates the unambiguous sequential linking of the backbone amino acid residues. The potential of sequential 1H data acquisition procedure in the study of RNA is also demonstrated here.

Solid-State NMR Spectra of Lipid-Anchored Proteins under Magic Angle Spinning

Solid-state NMR is a promising tool for elucidating membrane-related biological phenomena. We achieved the measurement of high-resolution solid-state NMR spectra for a lipid-anchored protein embedded in lipid bilayers under magic angle spinning (MAS). To date, solid-state NMR measurements of lipid-anchored proteins have not been accomplished due to the difficulty in supplying sufficient amount of stable isotope labeled samples in the overexpression of lipid-anchored proteins requiring complex posttranslational modification. We designed a pseudo lipid-anchored protein in which the protein component was expressed in E. coli and attached to a chemically synthesized lipid-anchor mimic. Using two types of membranes, liposomes and bicelles, we demonstrated different types of insertion procedures for lipid-anchored protein into membranes. In the liposome sample, we were able to observe the cross-polarization and the (13)C-(13)C chemical shift correlation spectra under MAS, indicating that the liposome sample can be used to analyze molecular interactions using dipolar-based NMR experiments. In contrast, the bicelle sample showed sufficient quality of spectra through scalar-based experiments. The relaxation times and protein-membrane interaction were capable of being analyzed in the bicelle sample. These results demonstrated the applicability of two types of sample system to elucidate the roles of lipid-anchors in regulating diverse biological phenomena.

The DQF-COSY NMR Experiment, a Way to Detect Small J Couplings in the Case of Fast Relaxing Nuclei: Application to <sup>59</sup>Co in the Tetrahedral Mixed-Metal Cluster HRuCo<sub>3</sub>(CO)<sub>11</sub>(PPh<sub>3</sub>)

The first investigation and analysis of 59Co 2D NMR homonuclear chemical shift correlation spectra for a ruthenium-based tetrahedral mixed-metal cluster HRuCo3(CO)11(PPh3) are reported. For this cluster in solution and by contrast to the conventional COSY NMR experiment, the double-quantum filtered (DQF) COSY NMR spectrum proves the existence of a scalar coupling constant between 59Co nuclei. In order to obtain a value of this coupling, the 2D COSY spectrum for a three-spin 7/2 AX2 system has been simulated by numerical density matrix calculations. The comparison between experimental and theoretical 2D NMR COSY spectra gives a spin-coupling constant |1J(59Co-59Co)| ＜ 300 Hz for this cluster.

Sequential protein NMR assignments in the liquid state via sequential data acquisition

Two different NMR pulse schemes involving sequential 1H data acquisition are presented for achieving protein backbone sequential resonance assignments: (i) acquisition of 3D {HCCNH and HNCACONH} and (ii) collection of 3D {HNCOCANH and HNCACONH} chemical shift correlation spectra using uniformly 13C,15N labelled proteins. The sequential acquisition of these spectra reduces the overall experimental time by a factor of ≈2 as compared to individual acquisitions. The suitability of this approach is experimentally demonstrated for the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus.

Resolution and measurement of heteronuclear dipolar couplings of a noncrystalline protein immobilized in a biological supramolecular assembly by proton-detected MAS solid-state NMR spectroscopy

Two-dimensional 15N chemical shift/1H chemical shift and three-dimensional 1H–15N dipolar coupling/15N chemical shift/1H chemical shift MAS solid-state NMR correlation spectra of the filamentous bacteriophage Pf1 major coat protein show single-site resolution in noncrystalline, intact-phage preparations. The high sensitivity and resolution result from 1H detection at 600MHz under 50kHz magic angle spinning using ∼0.5mg of perdeuterated and uniformly 15N-labeled protein in which the exchangeable amide sites are partially or completely back-exchanged (reprotonated). Notably, the heteronuclear 1H–15N dipolar coupling frequency dimension is shown to select among 15N resonances, which will be useful in structural studies of larger proteins where the resonances exhibit a high degree of overlap in multidimensional chemical shift correlation spectra.

Identifying inter-residue resonances in crowded 2D 13C–13C chemical shift correlation spectra of membrane proteins by solid-state MAS NMR difference spectroscopy

The feasibility of using difference spectroscopy, i.e. subtraction of two correlation spectra at different mixing times, for substantially enhanced resolution in crowded two-dimensional (13)C-(13)C chemical shift correlation spectra is presented. With the analyses of (13)C-(13)C spin diffusion in simple spin systems, difference spectroscopy is proposed to partially separate the spin diffusion resonances of relatively short intra-residue distances from the longer inter-residue distances, leading to a better identification of the inter-residue resonances. Here solid-state magic-angle-spinning NMR spectra of the full length M2 protein embedded in synthetic lipid bilayers have been used to illustrate the resolution enhancement in the difference spectra. The integral membrane M2 protein of Influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH and is essential for the viral lifecycle. Based on known amino acid resonance assignments from amino acid specific labeled samples of truncated M2 sequences or from time-consuming 3D experiments of uniformly labeled samples, some inter-residue resonances of the full length M2 protein can be identified in the difference spectra of uniformly (13)C labeled protein that are consistent with the high resolution structure of the M2 (22-62) protein (Sharma et al., Science 330(6003):509-512, 2010).

Protein Structure Determination with Paramagnetic Solid-State NMR Spectroscopy

Many structures of the proteins and protein assemblies that play central roles in fundamental biological processes and disease pathogenesis are not readily accessible via the conventional techniques of single-crystal X-ray diffraction and solution-state nuclear magnetic resonance (NMR). On the other hand, many of these challenging biological systems are suitable targets for atomic-level structural and dynamic analysis by magic-angle spinning (MAS) solid-state NMR spectroscopy, a technique that has far less stringent limitations on the molecular size and crystalline state. Over the past decade, major advances in instrumentation and methodology have prompted rapid growth in the field of biological solid-state NMR. However, despite this progress, one challenge for the elucidation of three-dimensional (3D) protein structures via conventional MAS NMR methods is the relative lack of long-distance data. Specifically, extracting unambiguous interatomic distance restraints larger than ∼5 Å from through-space magnetic dipole-dipole couplings among the protein (1)H, (13)C, and (15)N nuclei has proven to be a considerable challenge for researchers. It is possible to circumvent this problem by extending the structural studies to include several analogs of the protein of interest, intentionally modified to contain covalently attached paramagnetic tags at selected sites. In these paramagnetic proteins, the hyperfine couplings between the nuclei and unpaired electrons can manifest themselves in NMR spectra in the form of relaxation enhancements of the nuclear spins that depend on the electron-nucleus distance. These effects can be significant for nuclei located up to ∼20 Å away from the paramagnetic center. In this Account, we discuss MAS NMR structural studies of nitroxide and EDTA-Cu(2+) labeled variants of a model 56 amino acid globular protein, B1 immunoglobulin-binding domain of protein G (GB1), in the microcrystalline solid phase. We used a set of six EDTA-Cu(2+)-tagged GB1 mutants to rapidly determine the global protein fold in a de novo fashion. Remarkably, these studies required quantitative measurements of only approximately four or five backbone amide (15)N longitudinal paramagnetic relaxation enhancements per residue, in the complete absence of the usual internuclear distance restraints. Importantly, this paramagnetic solid-state NMR methodology is general and can be directly applied to larger proteins and protein complexes for which a significant fraction of the signals can be assigned in standard 2D and 3D MAS NMR chemical shift correlation spectra.

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC'C and 3D C'NCA with sequential (13)C acquisitions, 3D NHH and 3D NC'H with sequential (1)H acquisitions and 3D CANH and 3D C'NH with broadband (13)C-(15)N mixing are demonstrated using microcrystalline samples of the β1 immunoglobulin binding domain of protein G (GB1) and the chicken α-spectrin SH3 domain.