Multiple quadrature detection in reduced dimensionality experiments

A new, simple procedure is proposed which enables acquisition of two or more chemical shifts encoded in a common dimension simultaneously in quadrature. For n chemical shifts projected in a single dimension, the expected effect is obtained by interleaved acquisition and appropriate combination of 2ⁿ data sets per increment of respective evolution time. The particular chemical shifts can be calculated from sums and differences of signal frequencies obtained by different combination of the acquired data sets. In comparison to the established reduced dimensionality (RD) techniques, the proposed method enhances resolution due to reduction of the number of signals and requires less evolution time increments owing to narrower spectral width in the RD-domain. We show examples of the application of the new approach to the 2D HNCA and HN(CO)CA techniques with two, and 2D HACANH with three frequencies simultaneously encoded in the t ₁ evolution period, for ¹³C,¹⁵N-labeled ubiquitin.     

Sensitivity enhancement in (HCA)CONH experiments

A novel sensitivity-enhancement technique is proposed for experiments which correlate protein backbone resonances and start with magnetization from ¹³C-¹H groups. The technique is based on replenishing magnetization lost by dipole-CSA cross-correlated relaxation of the ¹³C spin with ¹³C steady state magnetization. The principle is demonstrated for the (HCA)CONH experiment, resulting in 1.6-fold sensitivity enhancement compared to the HN(CA)CO experiment. Furthermore, other versions of the (HCA)CONH experiment were evaluated, including a novel experiment with spin-locking of transverse ¹³C-¹H two-spin coherence, and a cross-correlation compensated (CA)CONH experiment which starts from ¹³C rather than ¹H magnetization.     

Computer assignment of the backbone resonances of labelled proteins using two-dimensional correlation experiments

Summary We present ALPS (Assignment for Labelled Protein Spectra), a flexible computer program for the automatic assignment of backbone NMR resonances of ¹⁵N/¹³C-labelled proteins. The program constructs pseudoresidues from peak-picking lists of a set of two-dimensional triple resonance experiments and uses either a systematic search or a simulated annealing-based optimization to perform the assignment. This method has been successfully tested on two-dimensional triple resonance spectra of Rhodobacter capsulatus ferrocytochrome c ₂ (116 amino acids).     

Heteronuclear correlation experiments for the determination of one-bond coupling constants

Spin-state selective experiments, HSQC-/ and CT-HMQC-/, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional,¹ H-detected ¹⁵N-¹H or¹³ C-¹H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra.     

Improved 4D NMR experiments for the assignment of backbone nuclei in13C/15N labelled proteins

Summary We recently proposed a novel 4D NMR strategy for the assignment of backbone nuclei in¹³C/¹⁵N-labelled proteins (Boucher et al., 1992). Intra-residue (and many sequential) assignments are obtained from a HCANNH experiment, whereas sequential assignments are based on a complementary HCA(CO)NNH experiment. We present here new constant time 4D HCANNH, HCA(CO)NNH and HNCAHA experiments that are more sensitive. Some of the data were presented at the 33rd ENC held at Asilomar, California, U.S.A., in April 1992.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

Efficient side-chain and backbone assignment in large proteins: Application to tGCN5

In determining the structure of large proteins by NMR, it would be desirable to obtain complete backbone, side-chain, and NOE assignments efficiently, with a minimum number of experiments and samples. Although new strategies have made backbone assignment highly efficient, side-chain assignment has remained more difficult. Faced with the task of assigning side-chains in a protein with poor relaxation properties, the Tetrahymena histone acetyltransferase tGCN5, we have developed an assignment strategy that would provide complete side-chain assignments in cases where fast ¹³C transverse relaxation causes HCCH-TOCSY experiments to fail. Using the strategy presented here, the majority of aliphatic side-chain proton and carbon resonances can be efficiently obtained using optimized H(CC-CO)NH-TOCSY and (H)C(C-CO)NH-TOCSY experiments on a partially deuterated protein sample. Assignments can be completed readily using additional information from a ¹³ C-dispersed NOESY-HSQC spectrum. Combination of these experiments with H(CC)NH-TOCSY and (H)C(C)NH-TOCSY may provide complete backbone and side-chain assignments for large proteins using only one or two samples.     

Four-dimensional heteronuclear correlation experiments for chemical shift assignment of solid proteins

Chemical shift assignment is the first step in all established protocols for structure determination of uniformly labeled proteins by NMR. The explosive growth in recent years of magic-angle spinning (MAS) solid-state NMR (SSNMR) applications is largely attributable to improved methods for backbone and side-chain chemical shift correlation spectroscopy. However, the techniques developed so far have been applied primarily to proteins in the size range of 5–10 kDa, despite the fact that SSNMR has no inherent molecular weight limits. Rather, the degeneracy inherent to many 2D and 3D SSNMR spectra of larger proteins has prevented complete unambiguous chemical shift assignment. Here we demonstrate the implementation of 4D backbone chemical shift correlation experiments for assignment of solid proteins. The experiments greatly reduce spectral degeneracy at a modest cost in sensitivity, which is accurately described by theory. We consider several possible implementations and investigate the CANCOCX pulse sequence in detail. This experiment involves three cross polarization steps, from H to CA[i], CA[i] to N[i], and N[i] to C′[i−1], followed by a final homonuclear mixing period. With short homonuclear mixing times (<20 ms), backbone correlations are observed with high sensitivity; with longer mixing times (>200 ms), long-range correlations are revealed. For example, a single 4D experiment with 225 ms homonuclear mixing time reveals ∼200 uniquely resolved medium and long-range correlations in the 56-residue protein GB1. In addition to experimental demonstrations in the 56-residue protein GB1, we present a theoretical analysis of anticipated improvements in resolution for much larger proteins and compare these results in detail with the experiments, finding good agreement between experiment and theory under conditions of stable instrumental performance.     

A doublet-separated sensitivity-enhanced HSQC for the determination of scalar and dipolar one-bond J-couplings

A simple, sensitivity-enhanced HSQC experiment is described which separates the upfield and downfield components in the indirect dimension into different subspectra. The sequence is similar to the generalized TROSY scheme; however, decoupling of the X-nucleus is used during detection. A detailed analysis of relaxation effects, precision and sensitivity of the method is presented. The approach is demonstrated in a two-dimensional water flip-back 1H- 15N HSQC which measures 1JHN splittings in isotropic and oriented samples of ubiquitin and the hepatitis C protease. The results are in excellent agreement with splittings obtained from a conventional 1H-coupled HSQC.     

Half-filter experiments for assignment, structure determination and hydration analysis of unlabelled ligands bound to 13C/15N labelled proteins

A novel variant of the 13C/15N 2 half-filter experiment is reported for studying the hydration of an unlabelled ligand bound to a 15N and 13C uniformly labelled biological macromolecule. This doubly tuned filter experiment represents a powerful tool for obtaining resonance assignments, structure determination and hydration properties of a ligand. Its application to the binary complex formed by the inserted-domain (I-domain) of the leukocyte function-associated antigen-1 (LFA-1) with a ligand reveals the presence of H2O molecules at the binding interface.     

A novel protocol based on HN(C)N for rapid resonance assignment in ((15)N, (13)C) labeled proteins: implications to structural genomics

A novel protocol, based on the HN(C)N experiment, has been developed for rapid assignment of backbone H(N) and (15)N resonances in ((15)N, (13)C) labeled proteins. The protocol exploits the directly observable (15)N and H(N) sequential correlations and the distinctive peak patterns in the different planes of the HN(C)N spectrum, depending upon the nature of the residues displaying the correlations. Glycines and prolines, which are responsible for the distinctive features, provide many check/start points for the sequential walks. These features enhance the speed of data analysis and render side chain assignments less crucial for the success of the assignments. The application of the protocol has been demonstrated with FK506 binding protein (FKBP, molecular mass 12 kDa).     

Optimized set of two-dimensional experiments for fast sequential assignment,secondary structure determination,and backbone fold validation of 13C/15N-labelled proteins

NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional ¹H-¹⁵N HSQC-like spectra. The ¹H-¹⁵N correlation peaks are frequency shifted by an amount ± _X along the ¹⁵N dimension, where _X is the C, C, or H frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces.     

Spectrophotometric determination of formation constants for the Cu-ethylenediamine-halogen (chloride and bromide) system and their catalytic effect on the oxidative coupling of 2,6-di-tert-butyl-phenol

In order to explain the mechanism of the dimerization of 2,6-di-tert-butyl-phenol when catalyzed by the copper-ethylenediamine complexes, a spectrophotometric study of the speciation of copper(II) complexes in methanol of Cu(II), ethylendiamine and Cl⁻ or Br⁻ was carried out at 303 K. The formation constants obtained for the copper chloride system are: log β₁₀₁ = 2.90 ± 0.03, log β₁₀₂ = 6.39 ± 0.03 and log β₁₀₃ = 8.62 ± 0.04, for the copper bromide system are log β₁₀₁ = 3.01 ± 0.10, log β₁₀₂ = 5.50 ± 0.08, for the copper-ethylendiamine complexes are log β₁₁₀ = 6.13 ± 0.05 and log β₁₂₀ = 10.54 ± 0.08, and for the ternary copper-ethylenediamine chloride or bromide systems are log β₁₁₁ = 10.21 ± 0.03 and log β₁₁₁ = 10.07 ± 0.03, respectively. Knowing the speciation of the copper-ethylenediamine-halide systems, the kinetic studies can be correlated with the species in solution. Comparative studies of the oxidation reaction of 2,6-di-tert-butyl-phenol using different copper(II) complexes with chloride or bromide and ethylenediamine as catalyst are reported. Their catalytic activity in the oxidation of 2,6-di-tert-butyl-phenol was monitored in methanol solution, following the corresponding quinone formation, at 418 nm (ε = 3.95 × 10⁴ mol⁻¹ L cm⁻¹ at 303 K). The results indicate that the most active species are [Cu(en)X]⁺, where X is bromide or chloride, Both complexes have similar activity.     

An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated 15N, 13C-labeled proteins

Summary A biosynthetic strategy has recently been developed for the production of ¹⁵N, ¹³C, ²H-labeled proteins using ¹H₃C-pyruvate as the sole carbon source and D₂O as the solvent. The methyl groups of Ala, Val, Leu and Ile (2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a ¹⁵N, ¹³C, ²H, ¹H₃C-labeled SH2 domain at 3°C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30–40 kDa molecular weight range.     

Local propensities and statistical potentials of backbone dihedral angles in proteins

The following three issues concerning the backbone dihedral angles of protein structures are presented. (1) How do the dihedral angles of the 20 amino acids depend on the identity and conformation of their nearest residues? (2) To what extent are the native dihedral angles determined by local (dihedral) potentials? (3) How to build a knowledge-based potential for a residue's dihedral angles, considering the identity and conformation of its nearest residues? We find that the dihedral angle distribution for a residue can significantly depend on the identity and conformation of its adjacent residues. These correlations are in sharp contrast to the Flory isolated-pair hypothesis. Statistical potentials are built for all combinations of residue triplets and depend on the dihedral angles between consecutive residues. First, a low-resolution potential is obtained, which only differentiates between the main populated basins in the dihedral angle density plots. Minimization of the dihedral potential for 125 test proteins reveals that most native alpha-helical residues (89%) and a large fraction of native beta-sheet residues (47%) adopt conformations close to their native one. For native loop residues, the percentage is 48%. It is also found that this fraction is higher for residues away from the ends of alpha or beta secondary structure elements. In addition, a higher resolution potential is built as a function of dihedral angles by a smoothing procedure and continuous functions interpolations. Monte Carlo energy minimization with this potential results in a lower fraction for native beta-sheet residues. Nevertheless, because of the higher flexibility and entropy of beta structures, they could be preferred under the influence of non-local interactions. In general, most alpha-helices and many beta-sheets are strongly determined by the local potential, while the conformations in loops and near the end of beta-sheets are more influenced by non-local interactions.     

A new triple-resonance experiment for the sequential assignment of backbone resonances in proteins

Summary A new protocol is described for obtaining intraresidual and sequential correlations between carbonyl carbons and amide ¹H and ¹⁵N resonances of amino acids. Frequency labeling of ¹³CO spins occurs during a period required for the ¹³C-¹⁵N polarization transfer, leading to an optimized transfer efficiency. In a four-dimensional version of the experiment, ¹³C chemical shifts are used to improve the dispersion of signals. The resonance frequencies of all backbone nuclei can be detected in a 3D variant in which cross peaks are split along two frequency axes. This pulse scheme is the equivalent of a five-dimensional experiment. The novel pulse sequences are applied to flavodoxin from Desulfovibrio vulgaris.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.     

High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies

Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with significant pharmacological importance. An efficient and straightforward expression strategy that enables cost-effective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-effective expression system in Escherichia coli for the production of ET-1 enriched with (15)N and (13)C isotopes. Employing thioredoxin as carrier protein, specific and nearly quantitative cleavage of ET-1 from the fusion was mediated by Factor Xa, and purification to homogeneity (final purity of >95%) was achieved by RP-HPLC. Purified recombinant ET-1 was found to be indistinguishable from the synthetic counterpart as determined by mass spectrometry and NMR spectroscopy. Our expression strategy offers the potential for production of isotopically labeled ET-1 in large (mg) quantities for the purpose of heteronuclear NMR experiments. Moreover, the method devised should be applicable for recombinant expression of small peptides in general.     

Backbone resonance assignment in large protonated proteins using a combination of new 3D TROSY-HN(CA)HA, 4D TROSY-HACANH and 13C-detected HACACO experiments

A new method for backbone resonance assignment suitable for large proteins with the natural ¹H isotope content is proposed based on a combination of the most sensitive TROSY-type triple-resonance experiments. These techniques include TROSY-HNCO, ¹³C-detected 3D multiple-quantum HACACO and the newly developed 3D TROSY multiple-quantum-HN(CA)HA and 4D TROSY multiple-quantum-HACANH experiments. The favorable relaxation properties of the multiple-quantum coherences, signal detection using the ¹³C antiphase coherences, and the use of TROSY optimize the performance of the proposed set of experiments for application to large protonated proteins. The method is demonstrated with the 44 kDa uniformly ¹⁵N,¹³C-labeled and fractionally (35%) deuterated trimeric B. Subtilis Chorismate Mutase and is suitable for proteins with large correlation times but a relatively small number of residues, such as membrane proteins embedded in micelles or oligomeric proteins.