Chemical Shift Correlation Research Articles

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.

Quantitative Interpretation of Chemical Shifts Enables Mapping Proteins Conformational Landscape

To carry out their function, most proteins need to undergo transitions among different states. Understanding how the equilibrium between different states is altered upon ligand binding or interaction with other biological partners is of critical importance to explain protein function.Nuclear Magnetic Resonance isotropic 1H and 15N chemical shifts are a sensitive probe for both protein structure and conformational dynamics, and thus are an ideal observable to monitor equilibrium shifts. Yet, noise in the peak positions complicates the quantitative correlation of chemical shifts to the equilibrium position. Here we propose a robust methodology that relies on statistical analysis of NMR 1H/15N HSQC data and provides a quantitative measure of the equilibrium shift associated to ligand or substrate binding. In short, linearity in the 1H/15N chemical shifts position across different protein constructs is first identified by principal component analysis. Then, the scores of all residues on their principal components are filtered and combined together, and the shift in equilibrium position for each state is given in terms of confidence intervals. Finally, the degree of collectiveness of the protein response to ligand binding is mapped onto the protein structure by applying an adapted version of the chemical shifts covariance analysis (CHESCA). We illustrate the method in its applications to ligand binding to protein kinase A (PKA) and to a series of mutants of phospholamban. The results show that the method is capable of discerning the multiple states populated upon binding of different ligands to PKA as well as the shift in equilibrium position induced by mutations of phospholamban serine 16. Noticeably, the CHESCA analysis shows that while PKA response is highly collective throughout the whole protein, the PLN response is collective only in the surroundings of the perturbed region (domain 1A).

Finding Order in Disorder: Probing Transient Functional States in the Amyloidogenic Alzheimer's Aβ Peptide Using the NMR Chemical Shift Covariance Analysis (CHESCA)

Prior to self-association into toxic soluble oligomers, amyloidogenic peptides are often unstructured or only partially structured and they are best described by an heterogeneous conformational ensemble, in which multiple discrete structures are populated. Despite the complexity in the ensemble of conformations accessible to amyloidogenic peptides, it has been suggested that a key determinant of the propensity to aggregate is the transient access to a subset of functional conformations, i.e. conformers that are either aggregation competent or are incompetent but compete with aggregation competent states. However, due to the conformational ‘noise’ generated by the highly degenerate free-energy landscape typical of amyloidogenic peptides, the identification of minor populations of functional conformers relevant for aggregation remains experimentally challenging. Here, we propose a method to identify which residues within amyloidogenic peptides are involved in functional states based on the covariance analysis of NMR chemical shifts (CHESCA) [1]. CHESCA relies on linear correlations between residue-specific chemical shifts measured for the peptide system of interest subject to a set of perturbations that modulate a given functional property, e.g. the aggregation propensity in the case of amyloidogenic peptides. Such linear chemical shift correlations are useful to identify the residues involved in transient and minimally populated partially folded conformers. In addition, a distinct advantage of the chemical shift covariance method is that the relative order in which the perturbed states appear in the inter-residue correlations defines the functional relevance of the partially folded conformers. This general approach will be illustrated through its application to the Aβ peptide, which self-associates into insoluble β-sheet structured aggregates linked to Alzheimer's disease.1) Selvaratnam R, Chowdhury S, VanSchouwen B, Melacini G. Proc Natl Acad Sci U S A. 2011;108(15):6133.

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.

Theoretical predictions of the two-dimensional solid-state NMR spectra: A case study of the 13C–1H correlations in metergoline

A new method for the treatment of data from multidimensional solid-state NMR investigations is described. It approximates the theoretical NMR chemical shifts from the chemical shielding values obtained by first-principles calculations and subsequently treats these results to quantify the similarity between predicted and experimental chemical shift correlations. The test case of this approach is performed for the measured and several sets of computed 13C–1H heteronuclear correlations in the polymorphic form I of metergoline, which is relatively large, pharmaceutically active system. The proposed protocol is general, however, and it can be immediately applied to study other compounds and nuclei.

Correlation of substituted aromatic β‐diketones' characteristic protons chemical shifts with Hammett substituent constants

Influence of dibenzoylmethane's substituents in meta and para positions on chemical shift values of tautomers' characteristic protons was investigated in four solvents with (1)H NMR spectroscopy: acetone-d(6), benzene-d(6), CDCl(3) and deuterated dimethyl sulfoxide (DMSO-d(6)). It was proved that the influence of substituents on chemical shifts strongly depends on the kind of the solvent; the greatest changes were observed in benzene-d6 and the smallest in CDCl(3). In acetone-d(6) and DMSO-d(6), the influence of substituents on chemical shifts is similar and the most regular. It allowed a fair correlation of chemical shifts of para-substituted dibenzoylmethane derivatives' characteristic protons with Hammett substituent constants in these solvents. In CDCl(3), characteristic protons' chemical shifts were near (1)H NMR spectroscopy measurement error limits, and, therefore, correlation with Hammett substituent constants in this solvent was unsatisfactory. In benzene, although the changes of chemical shifts are the most evident, the changes are also the most irregular, and, therefore, correlation in this solvent failed completely. Results of meta-substituted derivatives were much more irregular, and their correlation with Hammett substituent constants was poor in all investigated solvents.

An Experimental and Theoretical Conformational Study of a Series of Substituted 3-cyclohexyl-2-phenyl-1,3-thiazolidin-4-ones

A series of novel 3-cyclohexyl-2-phenyl-1,3-thiazolidin-4-one derivatives with substituents on the 2-phenyl ring were synthesized, and a study of their solid state and solution conformations was effected. In solution the thiazolidin-4-one ring preferred the C2 phenyl in a pseudo-equatorial orientation and the solid state indicated a preference for the C2 phenyl being in a pseudo-axial orientation. Less robust substituent chemical shift (SCS) correlations utilizing 13C NMR were observed in this instance than in prior studies. Molecular modeling studies using Moller-Plessett second-order perturbation theory (MP2) indicate that the thiazolidinone ring and cyclohexyl ring conformations exhibit a global minimum consistent with the solution studies.

Hyperpolarized Hadamard Spectroscopy Using Flow NMR

The emergence of the dissolution dynamic nuclear polarization (D-DNP) technique provides an important breakthrough to overcome inherent sensitivity limitations in nuclear magnetic resonance (NMR) experiments. In dissolution DNP, only a small amount of frozen sample is polarized, dissolved, and injected into an NMR spectrometer. Although substantially enhanced NMR signals can be obtained, the single scan nature of this technique a priori impedes the use of correlation experiments, which represent some of the most powerful applications of NMR spectroscopy. Here, an alternative method for multiscan spectroscopy from D-DNP samples utilizing a flow NMR probe is described. Multiple hyperpolarized segments of sample are sequentially injected using a purpose designed device. Hadamard spectroscopy can then be applied for obtaining chemical shift correlation information even from a small number of scans. This capability is demonstrated with a four-scan data set for obtaining the [(13)C,(1)H] correlations in the test molecule 1-butanol. Because of the effects of spin-lattice relaxation and concentration gradients in the D-DNP experiment, the subtractive process for Hadamard reconstruction requires an additional step of intensity scaling. For this purpose, a reconstruction procedure was developed that uses entropy maximization and is robust with respect to noise and signal overlap. In a broader sense, the multiscan NMR as described here is amenable to various correlation NMR experiments, and increases the versatility of D-DNP in small-molecule characterization.

Exploiting the Synergy of Powder X-ray Diffraction and Solid-State NMR Spectroscopy in Structure Determination of Organic Molecular Solids

We report a strategy for structure determination of organic materials in which complete solid-state nuclear magnetic resonance (NMR) spectral data is utilized within the context of structure determination from powder X-ray diffraction (XRD) data. Following determination of the crystal structure from powder XRD data, first-principles density functional theory-based techniques within the GIPAW approach are exploited to calculate the solid-state NMR data for the structure, followed by careful scrutiny of the agreement with experimental solid-state NMR data. The successful application of this approach is demonstrated by structure determination of the 1:1 cocrystal of indomethacin and nicotinamide. The 1H and 13C chemical shifts calculated for the crystal structure determined from the powder XRD data are in excellent agreement with those measured experimentally, notably including the two-dimensional correlation of 1H and 13C chemical shifts for directly bonded 13C–1H moieties. The key feature of this combined approach is that the quality of the structure determined is assessed both against experimental powder XRD data and against experimental solid-state NMR data, thus providing a very robust validation of the veracity of the structure.

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.

Chemical Shift Correlations from Hyperpolarized NMR Using a Single SHOT

A significant challenge in realizing the promise of the dissolution dynamic nuclear polarization technique for signal enhancement in high-resolution NMR lies in the nonrenewability of the hyperpolarized spin state. This property prevents the application of traditional two-dimensional correlation spectroscopy, which relies on regeneration of spin polarization before each successive increment of the indirect dimension. Since correlation spectroscopy is one of the most important approaches for the identification and structural characterization of molecules by NMR, it is important to find easily applicable methods that circumvent this problem. Here, we introduce the application of scaling of heteronuclear couplings by optimal tracking (SHOT) to achieve this goal. SHOT decoupling pulses have been numerically optimized on the basis of optimal control algorithms to obtain chemical shift correlations in C-H groups, either by acquiring a single one-dimensional (13)C spectrum with (1)H off-resonance decoupling or vice versa. Vanillin, which contains a number of functional groups, was used as a test molecule, allowing the demonstration of SHOT decoupling tailored toward simplified and accurate data analysis. This strategy was demonstrated for two cases: First, a linear response to chemical shift offset in the correlated dimension was optimized. Second, a pulse with alternating linear responses in the correlated dimension was chosen as a goal to increase the sensitivity of the decoupling response to the chemical shift offset. In these measurements, error ranges of ±0.03 ppm for the indirectly determined (1)H chemical shifts and of ±0.4 ppm for the indirectly determined (13)C chemical shifts were found. In all cases, we show that chemical shift correlations can be obtained from information contained in a single scan, which maximizes the ratio of signal to stochastic noise. Furthermore, a comprehensive discussion of the robustness of the method toward nonideal conditions is included based on experimental and simulated data. Unique features of this technique include the abilities to control the accuracy of chemical shift determination in spectral regions of interest and to acquire such chemical shift correlations rapidly-the latter being of interest for potential application in real-time spectroscopy.

Spinning-rate encoded chemical shift correlations from rotational resonance solid-state NMR experiments

Structural measurements in magic-angle-spinning (MAS) solid-state NMR rely heavily on 13C–13C distance measurements. Broadbanded recoupling methods are used to generate many cross-peaks, but have complex polarization transfer mechanisms that limit the precision of distance constraints and can suffer from weak intensities for distant peaks due to relaxation, the broad distribution of polarization, as well as dipolar truncation. Frequency-selective methods that feature narrow-banded recoupling can reduce these effects. Indeed, rotational resonance (R2) experiments have found application in many different biological systems, where they have afforded improved precision and accuracy. Unfortunately, a highly selective transfer mechanism also leads to few cross-peaks in the resulting spectra, which complicates the extraction of multiple constraints. R2-width (R2W) measurements that scan a range of MAS rates to probe the R2 matching conditions of one or more sites can improve precision, and also permit multiple simultaneous distance measurements. However, multidimensional R2W can be very time-consuming. Here, we present an approach that facilitates the acquisition of 2D-like spectra based on a series of 1D R2W experiments, by taking advantage of the chemical shift information encoded in the MAS rates where matching occurs. This yields a more time-efficient experiment with many of the benefits of more conventional multidimensional R2W measurements. The obtained spectra reveal long-distance 13C–13C cross-peaks resulting from R2-mediated polarization transfer. This experiment also enables the efficient setup and targeted implementation of traditional R2 or R2W experiments. Analogous applications may extend to other variable-MAS and frequency-selective solid-state NMR experiments.

Long-Range Interactions in the Alpha Subunit of Tryptophan Synthase Help to Coordinate Ligand Binding, Catalysis, and Substrate Channeling

The α-subunit of tryptophan synthase (αTS) catalyzes the conversion of indole-3-glycerol phosphate to d-glyceraldehyde-3-phosphate and indole. We propose that allosteric networks intrinsic to αTS are modulated by the binding of the β-subunit to regulate αTS function. Understanding these long-range amino acid networks in αTS thus gives insight into the coordination of the two active sites within TS. In this study, we have used Ala residues as probes for structural and dynamic changes of αTS throughout its catalytic cycle, in the absence of the β-subunit. Projection analysis of the chemical shift changes by site-specific amino acid substitutions and ligand titrations indicates that αTS has three important conformational states: ligand-free, glyceraldehyde-3-phosphate-bound(like), and the active states. The amino acid networks within these conformations are different, as suggested by chemical shift correlation analysis. In particular, there are long-range connections, only in the active state, between Ala47, which reports on structural and dynamic changes associated with the general acid/base Glu49, and residues within the β2α2 loop, which contains the catalytically important Asp60 residue. These long-range interactions are likely important for coordinating chemical catalysis. In the free state, but not in the active state, there are connections between the β2α2 and β6α6 loops that likely help to coordinate substrate binding. Changes in the allosteric networks are also accompanied by protein dynamic changes. During catalytic turnover, the protein becomes more rigid on the millisecond timescale and the active-site dynamics are driven to a faster nanosecond timescale.

Hydrogen bond interaction and dynamics in PMMA/PVPh polymer blends as revealed by advanced solid-state NMR

The hydrogen bond interactions and dynamics of the poly (methyl methacrylate) (PMMA) and poly (4-vinyl phenol) (PVPh) polymer blends were studied by a variety of advanced solid-state NMR techniques. The possible hydrogen bond interactions between the carbonyl group of PMMA and hydroxyl group of PVPh were successfully elucidated by two-dimensional 1H–1H spin-exchange 13C–1H heteronuclear chemical-shift correlation (HETCOR) NMR experiment. The 13C 2D separation of undistorted powder patterns by effortless recoupling (SUPER) experiments combined with quantum chemical calculations for the theoretical predictions of chemical-shift anisotropy (CSA) parameters were utilized to investigate the intermolecular hydrogen-bonding interactions and molecular conformation of the blends. 13C–1H PISEMA (polarization inversion and spin-exchange at magic angle) experiments at different temperatures were used to reveal the heterogeneous dynamics resulting from the cooperative motion associated with the hydrogen bond interaction. PISEMA analysis shows that the motion of aromatic group in PVPh is affected by the rotating motions of methyl in PMMA.

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.

Database proton NMR chemical shifts for RNA signal assignment and validation

The Biological Magnetic Resonance Data Bank contains NMR chemical shift depositions for 132 RNAs and RNA-containing complexes. We have analyzed the 1H NMR chemical shifts reported for non-exchangeable protons of residues that reside within A-form helical regions of these RNAs. The analysis focused on the central base pair within a stretch of three adjacent base pairs (BP triplets), and included both Watson–Crick (WC; G:C, A:U) and G:U wobble pairs. Chemical shift values were included for all 43 possible WC-BP triplets, as well as 137 additional triplets that contain one or more G:U wobbles. Sequence-dependent chemical shift correlations were identified, including correlations involving terminating base pairs within the triplets and canonical and non-canonical structures adjacent to the BP triplets (i.e. bulges, loops, WC and non-WC BPs), despite the fact that the NMR data were obtained under different conditions of pH, buffer, ionic strength, and temperature. A computer program (RNAShifts) was developed that enables convenient comparison of RNA 1H NMR assignments with database predictions, which should facilitate future signal assignment/validation efforts and enable rapid identification of non-canonical RNA structures and RNA-ligand/protein interaction sites.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-012-9683-9) contains supplementary material, which is available to authorized users.