Triple resonance HNCCCH experiments for correlating exchangeable and nonexchangeable cytidine and uridine base protons in RNA

Summary A set of triple resonance experiments is presented, providing through-bond H₂N/HN to H6 connectivities in uridines and cytidines in ¹³C-/¹⁵N-labeled RNAs. These connectivities provide an important link between the sequential assignment pathways for the exchangeable and nonexchangeable proton resonances in nucleic acids. Both 2D and pseudo-3D HNCCCH experiments were applied to a 30-nucleotide lead-dependent ribozyme, known as the leadzyme. The HN to H6 connectivities for three uridines in the leadzyme were identified from one 2D H(NCCC)H experiment, and the H₂N to H6 connectivities were identified for seven of the eight cytidines from the combination of a 2D H(NCCC)H and a pseudo-3D H(NCC)CH experiment.     

Suppression of anti-TROSY lines in a sensitivity enhanced gradient selection TROSY scheme

A simple modification of the TROSY pulse transfer scheme, suggested by Yang and Kay [J. Biomol. NMR 13 (1999) 3–10], is proposed which results in the suppression of unwanted anti-TROSY lines without any extra loss in sensitivity. The higher sensitivity of this TROSY transfer scheme therefore becomes available for 2D [¹⁵N, ¹H] TROSY correlation and 3D/4D ¹⁵N separated NOESY type experiments where complete suppression of the broad anti-TROSY lines is essential.     

Direct amide <Superscript>15</Superscript>N to <Superscript>13</Superscript>C transfers for solid-state assignment experiments in deuterated proteins

The assignment of protein backbone and side-chain NMR chemical shifts is the first step towards the characterization of protein structure. The recent introduction of proton detection in combination with fast MAS has opened up novel opportunities for assignment experiments. However, typical 3D sequential-assignment experiments using proton detection under fast MAS lead to signal intensities much smaller than the theoretically expected ones due to the low transfer efficiency of some of the steps. Here, we present a selective 3D experiment for deuterated and (amide) proton back-exchanged proteins where polarization is directly transferred from backbone nitrogen to selected backbone or sidechain carbons. The proposed pulse sequence uses only ¹H–¹⁵N cross-polarization (CP) transfers, which are, for deuterated proteins, about 30% more efficient than ¹H–¹³C CP transfers, and employs a dipolar version of the INEPT experiment for N–C transfer. By avoiding H^N–C (H^N stands for amide protons) and C–C CP transfers, we could achieve higher selectivity and increased signal intensities compared to other pulse sequences containing long-range CP transfers. The REDOR transfer is designed with an additional selective π pulse, which enables the selective transfer of the polarization to the desired ¹³C spins.     

1H(C) and 1H(N) total NOE correlations in a single 3D NMR experiment. 15N and 13C time-sharing in t1 and t2 dimensions for simultaneous data acquisition

Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as ¹⁵N and ¹³C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous ¹³C and ¹⁵N spin labeling coherence in both t ₁ and t ₂ dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between ¹H_N and ¹H_C resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, ¹⁵N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.     

3d haro-Noesy-ch3nh and caro-Noesy-ch3nh Experiments for Double Labeled Proteins

Precision in the determination of the 3D structures of proteins by NMR depends on obtaining an adequate number of NOE restraints. Ambiguity in the assignment of NOE cross peaks between aromatic and other protons is an impediment to high quality structure determination. Two pulse sequences, 3D H_aro-NOESY-CH₃NH and 3D C_aro-NOESY-CH₃NH, based on a modification of a technique for simultaneous detection of ¹³C-¹H (of CH₃) and ¹⁵N-¹H correlations in one measurement, are proposed in the present work. These 3D experiments, which are optimized for resolution in the ¹³C and ¹⁵N dimensions, provide NOE information between aromatic protons and methyl or amide protons. CH₂ moieties are filtered out and the CH groups in aromatic rings are selected, allowing their NOE cross peaks to be unambiguously assigned. Unambiguous NOEs connecting aromatic and methyl or amide protons will provide important restraints for protein structure calculations.     

A simultaneous 15N,1H- and 13C,1H-HSQC with sensitivity enhancement and a heteronuclear gradient echo

Summary New pulse sequences are introduced and discussed that allow for simultaneous acquisition of ¹⁵N,¹H-and ¹³C,¹H-HSQC correlations for fully ¹³C,¹⁵N-labeled biomacromolecules in combination with hetero-nuclear gradient echoes and sensitivity enhancement. The pulse sequence experimentally found to be optimal can be used as a building block, especially in time-consuming multidimensional NMR experiments. Due to the excellent solvent suppression obtained by employing heteronuclear gradient echoes, which allows detection of resonances under the water resonance, it would be possible to record two sensitivity-enhanced 4D experiments simultaneously on one sample dissolved in H₂O, e.g. a 4D ¹³C,¹H-HSQC-NOESY-¹⁵N, ¹H/¹³C,¹H-HSQC.     

Assignment Strategies for Large Proteins by Magic-Angle Spinning NMR: The 21-kDa Disulfide-Bond-Forming Enzyme DsbA

We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to  ∼ 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-¹³C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-¹³C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional ¹³C-¹³C spectrum.     

Resonance assignment strategies for the analysis of nmr spectra of proteins

Determination of the high resolution solution structure of a protein using nuclear magnetic resonance (NMR) spectroscopy requires that resonances observed in the NMR spectra be unequivocally assigned to individual nuclei of the protein. With the advent of modern, two-dimensional NMR techniques arose methodologies for assigning the¹H resonances based on 2D, homonuclear¹H NMR experiments. These include the sequential assignment strategy and the main chain directed strategy. These basic strategies have been extended to include newer 3D homonuclear experiments and 2D and 3D heteronuclear resolved and edited methods. Most recently a novel, conceptually new approach to the problem has been introduced that relies on heteronuclear, multidimensional so-called triple resonance experiments for both backbone and sidechain resonance assignments in proteins. This article reviews the evolution of strategies for the assignment of resonances of proteins.     

A fast NMR method for resonance assignments: application to metabolomics

We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled (¹²C) or ¹³C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [¹³C, ¹H] HSQC–TOCSY, (2) 2D ¹H–¹H TOCSY and (3) 2D ¹³C–¹H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [¹³C, ¹H] HSQC–TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete ¹H and ¹³C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.     

Fast Assignment of <Superscript>15</Superscript>N-HSQC Peaks using High-Resolution 3D HNcocaNH Experiments with Non-Uniform Sampling

We describe an efficient NMR triple resonance approach for fast assignment of backbone amide resonance peaks in the ¹⁵N-HSQC spectrum. The exceptionally high resolutions achieved in the 3D HncocaNH and hNcocaNH experiments together with non-uniform sampling facilitate error-free sequential connection of backbone amides. Data required for the complete backbone amide assignment of the 56-residue protein GB1 domain were obtained in 14 h. Data analysis was vastly streamlined using a ‘backbone NH walk’ method to determine sequential connectivities without the need for ¹³C chemical shifts comparison. Amino acid residues in the sequentially connected NH chains are classified into two groups by a simple variation of the NMR pulse sequence, and the resulting ‘ZeBra’ stripe patterns are useful for mapping these chains to the protein sequence. In addition to resolving ambiguous assignments derived from conventional backbone experiments, this approach can be employed to rapidly assign small proteins or flexible regions in larger proteins, and to transfer assignments to mutant proteins or proteins in different ligand-binding states.     

Multiple frequency saturation pulses reduce CEST acquisition time for quantifying conformational exchange in biomolecules

Exchange between conformational states is required for biomolecular catalysis, allostery, and folding. A variety of NMR experiments have been developed to quantify motional regimes ranging from nanoseconds to seconds. In this work, we describe an approach to speed up the acquisition of chemical exchange saturation transfer (CEST) experiments that are commonly used to probe millisecond to second conformational exchange in proteins and nucleic acids. The standard approach is to obtain CEST datasets through the acquisition of a series of 2D correlation spectra where each experiment utilizes a single saturation frequency to ¹H, ¹⁵N or ¹³C. These pseudo 3D datasets are time consuming to collect and are further lengthened by reduced signal to noise stemming from the long saturation pulse. In this article, we show how usage of a multiple frequency saturation pulse (i.e., MF-CEST) changes the nature of data collection from series to parallel, and thus decreases the total acquisition time by an integer factor corresponding to the number of frequencies in the pulse. We demonstrate the applicability of MF-CEST on a Src homology 2 (SH2) domain from phospholipase Cγ and the secondary active transport protein EmrE as model systems by collecting ¹³C methyl and ¹⁵N backbone datasets. MF-CEST can also be extended to additional sites within proteins and nucleic acids. The only notable drawback of MF-CEST as applied to backbone ¹⁵N experiments occurs when a large chemical shift difference between the major and minor populations is present (typically greater than ~?8 ppm). In these cases, ambiguity may arise between the chemical shift of the minor population and the multiple frequency saturation pulse. Nevertheless, this drawback does not occur for methyl group MF-CEST experiments or in cases where somewhat smaller chemical shift differences occur are present.     

Recent progress in heteronuclear long-range NMR of complex carbohydrates: 3D H2BC and clean HMBC

The new NMR experiments 3D H2BC and clean HMBC are explored for challenging applications to a complex carbohydrate at natural abundance of ¹³C. The 3D H2BC experiment is crucial for sequential assignment as it yields heteronuclear one- and two-bond together with COSY correlations for the ¹H spins, all in a single spectrum with good resolution and non-informative diagonal-type peaks suppressed. Clean HMBC is a remedy for the ubiquitous problem of strong coupling induced one-bond correlation artifacts in HMBC spectra of carbohydrates. Both experiments work well for one of the largest carbohydrates whose structure has been determined by NMR, not least due to the enhanced resolution offered by the third dimension in 3D H2BC and the improved spectral quality due to artifact suppression in clean HMBC. Hence these new experiments set the scene to take advantage of the sensitivity boost achieved by the latest generation of cold probes for NMR structure determination of even larger and more complex carbohydrates in solution.     

Sensitivity improvement for correlations involving arginine side-chain Nε/Hε resonances in multi-dimensional NMR experiments using broadband 15N 180° pulses

Due to practical limitations in available ¹⁵N rf field strength, imperfections in ¹⁵N 180° pulses arising from off-resonance effects can result in significant sensitivity loss, even if the chemical shift offset is relatively small. Indeed, in multi-dimensional NMR experiments optimized for protein backbone amide groups, cross-peaks arising from the Arg guanidino ¹⁵Nε (~85 ppm) are highly attenuated by the presence of multiple INEPT transfer steps. To improve the sensitivity for correlations involving Arg Nε–Hε groups, we have incorporated ¹⁵N broadband 180° pulses into 3D ¹⁵N-separated NOE-HSQC and HNCACB experiments. Two ¹⁵N-WURST pulses incorporated at the INEPT transfer steps of the 3D ¹⁵N-separated NOE-HSQC pulse sequence resulted in a ~1.5-fold increase in sensitivity for the Arg Nε–Hε signals at 800 MHz. For the 3D HNCACB experiment, five ¹⁵N Abramovich-Vega pulses were incorporated for broadband inversion and refocusing, and the sensitivity of Arg¹Hε-¹⁵Nε-¹³Cγ/¹³Cδ correlation peaks was enhanced by a factor of ~1.7 at 500 MHz. These experiments eliminate the necessity for additional experiments to assign Arg ¹Hε and ¹⁵Nε resonances. In addition, the increased sensitivity afforded for the detection of NOE cross-peaks involving correlations with the ¹⁵Nε/¹Hε of Arg in 3D ¹⁵N-separated NOE experiments should prove to be very useful for structural analysis of interactions involving Arg side-chains.     

Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase

Rho GTPases have attracted considerable interest as signaling molecules due to their variety of functional roles in cells. Rnd1 is a relatively recently discovered Rho GTPase with no enzymatic activity against its bound GTP nucleotide, setting it apart from other family members. Research has revealed a critical role for Rnd1 not only in neurite outgrowth, dendrite development, axon guidance, but also in gastric cancer and in endothelial cells during inflammation. Structural information is crucial for understanding the mechanism that forms the basis for protein–protein interactions and functions, but until recently there were no reports of NMR studies directly on the Rnd1 protein. In this paper we report assignments for the majority of Rnd1 NMR resonances based on 2D and 3D NMR spectra. Rnd1 assignment was a challenging task, however, despite optimization strategies that have facilitated NMR studies of the protein (Cao and Buck in Small GTPase 2:295–304, 2012). Besides common triple-resonance experiments, 3D HNCA, 3D HN(CO)CA, 3D HNCO which are usually employed for sequence assignment, 3D NOESY experiments and specific labeling of 13 kinds of amino acids were also utilized to gain as many ¹H(N), ¹³C, and ¹⁵N resonances assignments as possible. For 170 cross peaks observed out of 183 possible mainchain N–H correlations in the ¹H–¹⁵N TROSY spectrum, backbone assignment was finally completed for 127 resonances. The secondary structure was then defined by chemical shifts and TALOS+ based on the assignments. The overall structure in solution compares well with that of Rnd1 in a crystal, except for two short segments, residues 77–83 and residues 127–131. Given that some features are shared among Rho GTPases, Rnd1 assignments are also compared with two other family members, Cdc42 and Rac1. The overall level of Rnd1 assignment is lower than for Cdc42 and Rac1, consistent with its lower stability and possibly increased internal dynamics. However, while the Rnd1 switch II region remained un-assigned, the switch I region could be more fully assigned compared to Cdc42 and Rac1. The NMR assignment and structure analysis reported here provides a robust basis for future study of the binding between Rnd1 and other proteins, as well as for further studies of the molecular function of this unusual GTPase.     

HNCA-TOCSY-CANH experiments with alternate <Superscript>13</Superscript>C-<Superscript>12</Superscript>C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in ¹H₂O, and use ¹H excitation and detection. These experiments require alternate ¹³C-¹²C labeling together with perdeuteration, which allows utilizing the small ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} scalar coupling that is otherwise masked by the stronger ¹J_CC couplings in uniformly ¹³C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the ^{1 3} \textC^a ^{ 1 3} {\text{C}}^{\alpha } of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the ¹⁵N-¹H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.     

Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H₂O/50%D₂O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a ¹³C′-coupled 2D ¹⁵N–¹H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH₂ groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly <Superscript>15</Superscript>N labeled integral membrane proteins in magnetically aligned lipid bilayers

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring ¹H-¹⁵N dipolar couplings (DC) and ¹⁵N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([¹H,¹⁵N]-SE-PISEMA-PDSD). The incorporation of 2D ¹⁵N/¹⁵N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the ¹⁵N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.     

5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion

Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of ¹³C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.     

GFT projection NMR spectroscopy for proteins in the solid state

Recording of four-dimensional (4D) spectra for proteins in the solid state has opened new avenues to obtain virtually complete resonance assignments and three-dimensional (3D) structures of proteins. As in solution state NMR, the sampling of three indirect dimensions leads per se to long minimal measurement time. Furthermore, artifact suppression in solid state NMR relies primarily on radio-frequency pulse phase cycling. For an n-step phase cycle, the minimal measurement times of both 3D and 4D spectra are increased n times. To tackle the associated ‘sampling problem’ and to avoid sampling limited data acquisition, solid state G-Matrix Fourier Transform (SS GFT) projection NMR is introduced to rapidly acquire 3D and 4D spectral information. Specifically, (4,3)D (HA)CANCOCX and (3,2)D (HACA)NCOCX were implemented and recorded for the 6 kDa protein GB1 within about 10% of the time required for acquiring the conventional congeners with the same maximal evolution times and spectral widths in the indirect dimensions. Spectral analysis was complemented by comparative analysis of expected spectral congestion in conventional and GFT NMR experiments, demonstrating that high spectral resolution of the GFT NMR experiments enables one to efficiently obtain nearly complete resonance assignments even for large proteins.